Direct DNA fingerprinting of Hdicobacter pyiori in dental plaque by PCR amplification and restriction analysis of urease A gene sequences

R J Owenl, A Hurtadol, N Banatvala*, R A Feldman2, J M Hardie3

‘National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT; ZDepartment of Epidemiology and Medical Statistics at QMW, London Hospital Medical College, Mile End Road, London El 4NS; aDepartment of Oral Microbiology, London Hospital Medical College, Turner Street, London El 2AD. UK

Summary

The human mouth may act as a reservoir for Helicobacter pylori infection. A novel DNA- based fingerprinting technique was developed to compare dental plaque extracts with gastric biopsy cultures for evidence of common strain types at the two sites within an individual. Diversity within a 200 bp region of the ureA gene from six unrelated strains was demonstrated by DNA sequencing with up to seven single base substitutions detected. The 361 bp ureA nested polymerase chain reaction (PCR) amplicons from DNA derived from dental plaque extracts and gastric cultures were compared by Mboll restric- tion endonuclease analysis. Restriction length polymorphisms (RFLPs) were evident within the ureA segment and three distinct fingerprints of three or four bands were found. Results of a comparison between samples from oral and gastric sites demonstrated matching H. pylori strain types in two individuals, whereas six other individuals had different strain types. The results indicated the PCR restriction endonuclease analysis method can be applied directly to dental plaque without the need for positive culture of H. pylori. It is a rapid method and has potential for monitoring persistent H. py/ori re- infection and for studies on its transmission within families.

Key words: PCR, Helicobacrer py/ori, dental plaque, DNA fingerprinting

Serodiagn. Immunother. Infect. Disease 1994, Vol. 6, 196-202, December

Introduction

Helicobacter pylori is a Gram-negative microaerophilic curved bacterium, which colonizes the human gastric mucosa causing an infection that develops slowly yet can persist for decades’. The presence of the organism is often associated with chronic active gastritis and it is widely regarded to be a co-factor both for the devel- opment of duodenal ulceration* and for an increased risk of gastric carcinoma3.

Received: 10 August 1994 Accepted: 14 August 1994 Correspondence and reprint requests to: RJ Owen, National Colleckon of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue. London NW9 5HT. UK 0 1994 Butterworth-Heinemann Ltd 0888-0786/94/040196-07

Even though serodiagnosed infection with H. pylori is widespread, the mode of transmission is not known. The fact that bacteria have on occasion been cultured from dental plaqueU as well as from faeces’ gives support to the idea of person-to-person transmission, yet the role of both faecal-oral and oral-oral spread is far from estab- lished. Because H. pylori is isolated only rarely from sites other than the human gastric mucosa, interest has focused increasingly on the use of the polymerase chain reaction (PCR) as a means of directly detecting H. pylori in saliva or dental plaqueSI’, and in faecesr2. Recently, we reported that 74% of gastric biopsy culture positive individuals and 58% of asymptomatic Bangladeshi schoolchildren were dental plaque PCR positive’“.

PCR provides information about the presence or absence of H. pyfori in samples but not on the strain

Owen et al.: Direct DNA fingerprinting of H. pylori 197

type. The aim of the present study was to develop a molecular method that would allow the direct deter- mination of a genomic DNA fingerprint from the PCR amplification product, so that gastric and oral coloniza- tion of the same individual could be compared to avoid the problems associated with culturing from the mouth. DNA diversity amongst H. pyfori is well estab- lishedlj-lh and polymorphisms within the urease (ure) gene set are of practical value for DNA-based finger- printing of H. pylori’7-ZL). We have determined variable base positions within an internal segment of ureA and have used these as the basis for detecting polymor- phisms within the PCR product by restriction digest analysis. The procedure has been applied to compare oral and gastric colonization by H. pylori in eight individuals with gastric disease. in order to obtain more information about the role of the mouth as a reservoir of infection.

Materials and methods

Bacterial strains and culture conditions

The four strains (test set) of H. pylori used in sequenc- ing studies originated from human gastric biopsies in Australia (NCTC 11637, the type strain), England (A685/91), Northern Ireland (A616/92) and Italy (A646/92). The strains were selected because they were from different geographical origins and were genomically diverse according to previous PCR-RFLP analyses of the 2.4 kbp urease A and urease B gene fragment ilJ.ll’.

Eight other strains (clinical set) were from gastric biopsies of patients with gastrointestinal symptoms attending an endoscopy clinic at the Royal London Hospital (LH). The strains were isolated by inocula- tion of biopsy material onto brain heart infusion agar (BHIA) containing 5% (v/v) horse blood and H. pylori selective supplement (Oxoid SR 147E) containing vancomycin 10 mg l-l, trimethoprim 5 mg l-1, cefsolidin 5 mg 1-l and amphotericin B 5 mg 1-l. Cultures were incubated for 3-5 days at 37” C in microaerobic gaseous conditions (5% OZ. 5% COZ, 2% H, and 88% NJ using a gas generating kit (Oxoid BR056A), or a variable atmosphere incubator (Don Whitley Scientific Ltd., Shipley, Yorks, UK). Colonies with the appear- ance of H. pylori were identified as such by Gram stain and production of urease, oxidase and catalase using standard methods?‘. Bacterial isolates were subcultured onto BHIA to check for purity and then preserved in 10% glycerol in nutrient broth (Oxoid, Basingstoke, UK) over liquid nitrogen (-196” C). Strains were recovered for DNA extraction on BHIA and incubated for 48 h at 37” C under microaerobic conditions.

Dental plaque samples

The dental plaque samples were collected from each of the above eight patients (LH set) by scraping material from both supragingival and subgingival tooth surfaces

with sterile toothpicks prior to endoscopic examina- tion, as reported previously’j. The samples were stored initially over liquid nitrogen, and then at -70” C before subsequent DNA extraction and PCR analysis. An additional dental plaque sample (LH 248) was included in the sequencing studies but the matching gastric biopsy culture was not available for comparison.

Extraction of genomic DNA

Bacterial genomic DNA from dental plaque and from pure cultures isolated from gastric biopsies was extracted by the cetyltrimethyl-ammonium bromide (CTAB) method according to the DNA miniprep protocol of Wilson?‘. The pellet of DNA was redis- solved in 50 ~1 of TE (10 mM Tris and 1 mM EDTA pH 8.0).

Synthetic oligonucleotide primers for PCR of ureA

Oligonucleotide primers for PCR amplification of ureA internal regions were synthesized commercially (Pharmacia Biotec, Milton Keynes, UK). The 18 mer primers for the amplification of the urease A gene internal 411 bp fragment had the sequences 5’- GCCAATGGTAAATTAGTT-3’ (forward, position 360) and 5’-CTCCTTAATTGTTTTTAC-3’ (reverse. position 770) respectively as described by Clayton et al.lx. The two 20 mer oligomers from the urease A gene used as the nested inner primers were 5’-AGTTC- CTGGTGAGTTGT’I’CT-3’ (forward. position 374) and 5’-AGCGCCATGAAAACCACGCT-3’ (reverse. position 734) as described by Wang et al.‘“. After dilution in TE to a concentration of 10 FM, aliquots of each primer were stored at -20” C.

PCR amplification of ureA gene sequences

PCR was performed in a final volume of 100 ~1 of reaction mixture containing 10 mM Tris-HCI (pH 8.3). 50 mM KCl, 1.5 mM MgCl,, 0.01% (,w/v) gelatin, 0.05 mM each deoxynucleotide. 0.2 PM each primer and 2.5 units Thermus aquaticus (Taq) polymerase (Boehringer Mannheim, Lewes. UK). The template DNA for the standard PCR amplification consisted of 1 pl of the suspension after DNA extraction from each culture of H. pyfori and 10 ~1 from each dental plaque sample obtained as previously described’j. For the nested PCR amplification, which was necessary to obtain sufficient product for RFLP analyses, 1 ~1 of the standard amplification product was used as template. Each of the reaction mixtures were overlayed with 50 ~1 of mineral oil to prevent evapo- ration and PCR amplification was performed with an OmniGene automatic thermal cycler (Hybaid Ltd., Teddington, UK). The amplification cycle for the urease A gene internal 411 bp fragment consisted of denaturation at 94” C for 30 s, primer annealing at 48” C for 1 min and extension at 72” C for 1 min 30 s. The amplification cycle for the nested PCR fragment

198 Serodiagn. Immunother. Infect. Disease 1994; 6: No 4

involved denaturation at 94” C for 30 s, primer annealing at 56” C for 1 min and extension at 72” C for 1 min 15 s. In both cases an initial denaturation of the target DNA at 95” C for 5 min preceded the 30 consecutive cycles as described above. A final step at 72” C for 5 min was performed to ensure full exten- sion of the product. The product from each amplifi- cation reaction was analysed by electrophoresis of a 10 ~1 aliquot through 1.0 (w/v) agarose gels and the DNA bands were visualized by staining with ethidium bromide and excitation under UV light on a transil- luminator.

Restriction endonuclease analysis

The DNA product from each nested PCR amplifica- tion was ethanol precipitated and resuspended in a 2.5 ~1 TE. The DNA was added to 5.5 ~1 of sterile distilled water, 0.5 pl of spermidine, 1 pl of the appropriate buffer and 0.5 ~1 of Mb011 [GAAGA (N),] giving a final volume of 10 ~1. The amplicon for NCTC 11637 was also analysed with two additional restriction endonucleases, A/u1 and DdeI. The digest mixture was incubated at 37” C for 4 h according to the manufac- turer’s instructions (Gibco-BRL, Paisley, UK). The digestion of the PCR amplification product was stopped with 5 ~1 of stop-mix solution, then 3 ~1 of the digest was analysed by discontinuous sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE). Gels were cast to allow for 1 cm stacking gel and the final polyacrylamide concentrations were 20% (v/v) for the separation gel and 5% (v/v) for the stack- ing gel. Electrophoresis was carried out using the SE 260 dual cooled vertical slab gel electrophoresis unit (Hoefer Scientific Instruments, San Francisco, USA) for 45 min at a constant current of 30 mA. These conditions were used to facilitate resolution of small (cl00 bp) fragments. The DNA fragments were visual- ized by staining with ethidium bromide and excitation under UV light on a transilluminator.

DNA sequencing

The double-stranded PCR amplicons from purified DNA from four strains (test set) of H. pyfori and one dental plaque sample (LH 248) were analysed by agarose gel electrophoresis. A 10 ~1 aliquot was electrophoresed through 1% (w/v) agarose gels and then purified by electroelution to remove the residual primers and deoxynucleotide triphosphates. The 5’ ends of both sequencing primers (HPU-1 and HPU-2) were ?zP labelled by mixing 10 pmol of primer DNA, 2 ~1 of 10x polynucleotide kinase buffer (0.5 M Tris-HCl pH 7.6, 0.1 M MgCl,, 50 mM dithiothreitol, 1 mM spermidine HCl and 1 mM EDTA, pH S), 2 ~1 of y”*P dATP 10 pCi (2.5-5 ~1; 5000 Ci mmolF), 5 units of polynucleotide kinase (30 U ~1-1, NBL) and distilled water to a total volume of 20 ~1. The reaction mixture was incubated at 37” C for 30 min and stored at -20”

C. PCR products purified by the method of Kusukawa et al.?” were subjected to dideoxy chain termination sequencing with a single primer using the TAQuence DNA sequencing kit (USB, Amersham, Little Chalfont, UK) following the method of Embley*“. The sequencing reaction products were analysed on standard wedge-shaped sequencing gels (Pharmacia Biotec Macrophor). Gels were fixed using methanol/acetic acid/glycerol (lo%, lo%, 2% v/v), dried at 80” C and autoradiographed using Amersham Hyperfilm for 24-72 h. The sequences of the above amplicons were recorded and analysed on DNASTAR LaserGene 2000 software and compared with similar regions within the previously determined ureA sequences of Clayton et al.lx (GenBank accession number X17079) and Labigne et a1.2h (GenBank acces- sion number X57132).

Results

Sequencing of the ureA internal region

A 411 bp fragment internal to the urease A gene of DNA from each of the four strains (test set) of H. pylori and from the single dental plaque sample (LH 248) was obtained by standard PCR amplification. Approximately 200 bases within this ureA gene ampli- con were sequenced for each of the five samples, with the two sequencing primers giving an overlap of 158 bases (ureA positions 516-673). Seven positions with differing bases were identified in ureA sequences of the five samples (Figure 1). A further five nucleotide differences were identified when these five sequences were compared with the homologous region within the two published ureA sequences18J6. All seven sequences were different except for NCTC 11637 and dental plaque sample LH 248. Differences in nucleotide positions outside the overlapping 158 bp region of ureA were not utilized in the present analysis because sequencing was not performed in both directions for that region.

Figure 1. Nucleotide sequence homologies of the internal segment of the ureA gene from different strains of H. pyfori. Mboll cutting sites are underlined. The strain source of each sequence is given on the right hand side.

192 128

88

Profile: A B A B

Figure 2. Mboll restriction digest patterns of the 361 bp nested PCR product from amplified ureA of the following strains of H. pylori: lane 1, A616/92; lane 2, A685/91; lane 3, A646192; lane 4, NCTC 11637; lane 5, size marker (IO bp ladder; Gibco-BRL).

246

123

bP

Figure 3. Multiple restriction endonuclease digests of the ureA 361 bp nested PCR product from NCTC 11637 H. pylori. Lane 1, Mboll and A/d; lane 2, Mboll and Ddel; lane 3, Mboll, Ddel and Alul; lane 4, size markers (123 bp ladder; Gibco-BRL).

The 361 bp llreA nested PCR amplicons from the four strains (NCTC 11637, A616/92. A685/91 and A646/92) used in the above DNA sequencing study were analysed by digestion with MhoII. That particular endonuclease was selected because analysis of the DNA sequencing results indicated at least two restric- tion sites within the amplified region. Restriction analysis yielded two to three bands (Figure 2) depend- ing on the strain with two distinct patterns comprising

Owen et al.: Direct DNA fingerprinting of H. pylori 199

Table 1. PCR-RFLP fingerprinting results on H. pylori ureA amplicons from stomach and mouth samples of patients attending endoscopy clinics at the Royal London Hospital

Patient reference H. pylori PCR-RFLP pattern rype

Gastric biopsy Dental plaque

LH 98 A B LH 248 A B LH 362 B B LH 364 B B LH 366 A B LH 368 C B LH 415 B A LH 551 B A

LH, London Hospital. Mboll band patterns were as follows: pattern A with 41, 40, 88, 192 bp bands; pattern E with 41, 128, 192 bp; and pattern C with 128, 233 bp bands.

either 40 + 41 (doublet). 88 and 192 bp bands (type A; characteristic of A616/92 and A646/92). or 41, 128 and 192 bp bands (type B: characteristic of NCTC 11637 and A685/91). The 40 and 41 bp fragments appeared as faint bands in most photographs. Inspection of the DNA sequences on computer showed that the two patterns of three or four bands depended on the presence of adenine (A) or guanine (G) in lrreA position 635 (273 within the 361 bp fragment). A search of the published ureA sequences’x.‘h indicated MboII restriction sites that would yield 40, 41, 88 and 192 bp fragments characteristic of pattern type A. The dental plaque sample (LH 248) was not included in the PCR-RFLP analysis because insufficient material was available.

The amplicon from NCTC 11637 was investigated, to determine if a combination of endonucleases would give additional fragments to enhance the discrimina- tion of the PCR-RFLP analysis. Digests were made with MboII in combination with either AlzlI or oneI, or with all three enzymes simultaneously. The patterns obtained (Figure 3) consisted of just one or two fragments that were resolvable under the electrophore- sis conditions used. No other amplicons were investi- gated because the methcd appeared unlikely to offer improved discrimination between samples.

RFL P annl~vsis c!f ureA arnplicons ,front patient scwnples

The eight patient samples, which consisted of ampli- cons obtained by IrreA nested PCR from purified DNA from cultures of H. pylori originating from gastric biopsies and from direct PCR of matched dental plaque DNA extracts, were analysed by MhoII diges- tion. The RFLP patterns obtained are listed in Table 1. Overall three different patterns were detected. Two of these were identical to the A and B patterns defined above for the H. pjdori test set as well as an additional pattern designated as pattern C with two fragments of

200 Serodiagn. Immunother. Infect. Disease 1994; 6: No 4

128 and 233 bp. The frequency of the strains with the pattern types was 3 out of 8 for type A, 4 out of 8 for type B and 1 out of 8 for type C. By contrast, most (6 out of 8) of the dental plaque samples were type A and only two were type B. The PCR-RFLP patterns matched for just two patients (LH 362 and LH 344), whereas all the other patient samples had different patterns for H. pylori in the respective mouth and stomach paired samples.

Discussion

Our results on ureA PCR-RFLP analysis of samples from a series of eight patients showed that most (75%; 6 out of 8) individuals were apparently colonized in the mouth (dental plaque) by H. pylori that were genotyp- ically different from those in the antrum (culture). However, two individuals had H. pyfori with matching DNA fingerprints at each site. These latter results agree with the findings of Shames et al.17 who isolated one dental plaque strain with an identical genomic DNA restriction digest profile to the paired strain cultured from the stomach. Likewise, Khandaker et al.5 demonstrated that paired strains of H. pylori from the mouth (dental plaque cultures but not saliva) and antrum from each of five ulcer patients were identical by DNA fingerprinting (ribotyping). It is evident from these studies that different individuals may have either matching or different strain types present in the mouth and gastroduodenum at any given time. Mixed strain types are uncommon in the stomach2*J9 and it was not possible to determine from the present study if mixed strains were present in dental plaque.

Our use of DNA-based fingerprinting of PCR ampli- cons has enabled direct comparisons to be made between H. pylori from different sites, despite the diffi- culties associated with direct culturing from dental plaque. Cultures of H. pylori from the mouth are relatively rare when compared with the high recovery rates of H. pyfori from antral and duodenal biopsies. Even so, PCR amplification assays based mainly on the ribosomal RNA and urease genes, have detected H. pylori at much higher frequencies in dental plaque of both symptomatic and asymptomatic individuals%-[‘,‘“. From such evidence, H. pyZori would appear to be present in the mouth of many individuals but at levels too low for successful culturing in the presence of the large number of other organisms known to be present in dental plaque.

DNA sequencing of a 200 bp internal region of the urease A gene from a set of four diverse isolates of H. pylori enabled us to establish that there was sufficient allelic variation within the targetted region to provide a basis for fingerprinting isolates and sample extracts. Each strain differed in several single base mutations and they were different from the two previously published H. pylori ureA sequences’8,26. The predicted cutting sites that would likely result in the DNA fragments described here are shown in Figure 4. These sequencing results were consistent with other data

a 41 192 40 88 I

374 414 606 646 734

b _---

t-

---- - 41 192 40 88

-II ---

= rri:: 192 1 -__--I 128

Figure 4. Graphic representation of Mboll predicted cleavage sites within the H. pytori 361 bp nested PCR product from ureA (positions 374-734). a, Published sequences18ez6; b, A616142 and A646/91; c, NCTC 11637 and A685/91. - Region sequenced in this study; --- additional sequence from Clayton et a1.18. Sizes of’fragments are given in bp.

demonstrating diversity within the urease gene cluster as well as in other regions of the H. pyfori genomell.lS.iY. We also showed it was possible to sequence the ureA amplicon directly from a dental plaque sample and that its sequence was homologous with the same region from the H. pylori type strain (NCTC 11637).

As DNA sequencing is time consuming and expen- sive for investigating numbers of samples, we devel- oped a procedure for restriction endonuclease digest analysis of the H. pylori ureA amplicon so that samples could be fingerprinted quickly. However, two problems were encountered in this approach. First, a single PCR amplification yielded insufficient quantities of the 421 bp product for digest analysis and it was necessary to carry out a nested PCR that gave suffi- cient product but resulted in a smaller 361 bp ampli- con. Second, it was necessary to select an endonuclease for the PCR-RFLF analysis that could be predicted to have sufficient cutting sites within the nested PCR amplicon to allow fine discrimination between samples. Mb011 was selected from analysis of the available ureA sequences and when applied to the test set of strains, three different band patterns were obtained. There was predictably an unavoidable loss in discrimination when RFLP analysis was used instead of DNA sequencing. For the samples from the Royal London Hospital patients selected for study, discrimination was not a problem because most paired samples had different Mb011 PCR-RFLP fingerprints and could reliably be assumed to be different. However, for two patients (LH 362 and LH 364), the gastric biopsy and dental plaque samples had identical fingerprints, which suggested their ureA sequences were either truly identical or simply appeared to be so because any base mutations or substitutions in the region analysed were not affecting the Mb011 recognition sequences - as in the case of some samples in the test set (e.g. A616/92 and A646182).

DNA fingerprint discrimination can be enhanced by using additional endonucleases that sample other areas within the amplico@. Therefore we also tested two other endonucleases (D&I and AU) in combination

with MboII on the nested PCR product from NCTC 11637. These enzymes were selected because their digest conditions were compatible with MboII and they were predicted to have cutting sites within the ampli- con. Just one or two bands were detected and so there was no convincing evidence that such multiple enzyme digests would give additional discrimination between samples in this study. The smaller fragments predicted from such multiple enzyme digestions (~40 bp) were difficult to detect under the electrophoresis conditions used. However, the approach is being further investi- gated on other PCR samples as a possible way of increasing the discriminatory power of PCR-RFLP analysis. particularly for those samples that appear identical in MhoIl digest analysis alone.

We conclude from this study that PCR-RFLP analy- sis of rtreA amplicons provides a novel approach to determine if individuals have similar strains of H.

pyfori colonizing the dental plaque as well as the stomach, even when it was not possible to obtain pure cultures from the dental plaque. Application of this technique in the future using more discriminatory PCR-RFLP analysis or automated DNA sequencing, could provide important information about the role of the mouth as a reservoir for re-infection after treat- ment and in investigating the frequency of person-to- person spread of I-I. pvlori within families or communities.

Acknowledgements

Support for this work was provided in part by a grant from The Special Trustees, The Royal London Hospital. AH was the recipient of a training fellowship under the COMETT programme of the European Commission. We thank Dennis Linton for assistance with DNA sequencing and Louisa Clements and Y. Abdi for collecting the samples.

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