Development of a Scheme for Genotyping Helicobacter pylori Based on Allelic Variation in Urease Subunit Genes

Abstract

Helicobacter pylori urease subunit genes in 383 isolates from 10 countries were investigated by PCR-restriction fragment length polymorphism (HaeIII) analysis. Eighty-two different ureAB profiles were documented by reference to known sequences. Variation among 51% of strains was accounted for by 10 predominant patterns, which provided a unique framework for categorizing isolates with geographically diverse origins.

Helicobacter pylori is the causative agent worldwide of chronic gastritis and is a significant risk factor in the development of peptic ulceration and gastric cancer (5). Intense urease activity is a key pathogenicity factor (12), and in urease, the first genomic region to be sequenced, genes are highly conserved (2, 13, 28). Because DNA restriction digest analysis of isolates from different patients indicates a high level of intragenic diversity, the approach has been applied widely for genotyping (1, 2, 6, 7, 10, 15, 17, 18, 23–27). In view of the importance of urease as a candidate vaccine (14) and as a target for PCR specific-detection assays (15, 19), we have developed a scheme for indexing ureAB variation to facilitate identification of clinical isolates.

The 383 isolates of H. pylori were collected between 1992 and 1997 from gastric or duodenal biopsy tissue taken during routine endoscopy of patients presenting with symptoms of dyspepsia (10, 11, 18) or from the gastric juice of healthy volunteers (21). Three reference cultures (NCTC 11637, NCTC 11638, and NCTC 12455 strain 26695) were included. Isolates were mostly from United Kingdom (n = 323), but strains from nine other countries (Australia [3 isolates], Canada [8 isolates], China [1 isolate], Italy [4 isolates], Nigeria [7 isolates], Peru [2 isolates], South Africa [4 isolates], Turkey [26 isolates] and the United States [5 isolates]) were also included in the study to determine geographical diversity. Strains were cultured on 10% (vol/vol) Columbia blood agar at 37°C in a variable atmosphere incubator (Don Whitley Scientific Ltd., Shipley, United Kingdom) under microaerobic conditions (5% O₂, 5% CO₂, 2% H₂, and 88% N₂) and preserved at −196°C. Genomic DNA was extracted, the ureAB region was amplified, and restriction fragment length polymorphism (RFLP) analysis was performed as described previously (11, 18, 23). DNA fragment sizes were estimated from migration distances by using polynomial curve-fitting functions (16). Profiles were recorded in Microsoft Excel as derived fragment sizes (in base pairs) and analyzed as numerical strings according to their positions in the amplicon (Table 1). A novel Visual Basic macro (details are available from the corresponding author) was written to calculate genotype frequencies and to search for matches and partial matches with existing database profiles (9).

TABLE 1.

Details of the 10 most common H. pylori ureAB profiles

Profile	Band sizesa (bp)	Frequencyb	Reference strain	Other designations
uAB1	315, 457, 444, 678, 515	25	NCTC 13085	60190, ATCC 49503
uAB2	315, 457, 444, 383, 295, 515	18	NCTC 11637c	RPH 13487, CCUG 17874, ATCC 43504
uAB3	315, 457, 1,122, 515	23	NCTC 12455	26695d
uAB4	772, 1,059, [63], 515	14	NCTC 12385
uAB5	772, 827, 232, [63], 515	12	NCTC 13082
uAB6	772, 827, 295, 515	7	NCTC 13089
uAB10	315, 457, 827, 295, 515	11	NCTC 11638	RPH 13491, CCUG 17875
uAB11	315, 901, 615, [63], 515	9	NCTC 13090
uAB19	315, 457, 444, 165, 450, [63], 515	7	NCTC 13088
uAB32	772, 444, 678, 515	6	NCTC 13094

^aBands are ordered from 5′ to 3′ ends according to predicted locations of HaeIII sites within known ureA and ureB gene sequences (3, 13, 28). The 63-bp fragment (in brackets) was not detected in the RFLP analysis. 

^bBased on the numbers of infected patients and excluding multiple isolates from individual patients. 

^cType strain of species. 

^dGenome sequence has been published (28). 

The 2.41-kb ureAB amplicon was obtained from all 383 isolates of H. pylori. A total of 82 ureAB HaeIII RFLP patterns were defined and designated by a ureAB (uAB) profile number (uAB1 to uAB82). Examples of typical profiles are shown elsewhere (18). About 18% of the strains had unique uAB profiles, but the 10 most common profiles (Table 1) accounted for about 51% of strains. Profile descriptions were based on 17 fragments of ≤1,578 bp. The 515-bp fragment was conserved, but a scheme using seven diagnostic signature fragments for the rapid identification of the main profiles was devised (Table 2). The precise size and order of fragments in each profile were determined by using the known locations of HaeIII sites in linear maps of three previously reported ureAB sequences (3, 13, 28). NCTC 12455 (strain 26695) had profile uAB3, which was in accord with the HaeIII digest fragments predicted from the genome sequence (28). The estimated locations of the genetic events involving HaeIII sites are listed in Table 3. Pattern uAB1 was selected as the core pattern, as it was the most frequent and gave rise to the other common patterns by a series of single or double mutations. The 315-bp region at the 5′ end of ureA and the 515-bp region at the 3′ end of ureB were highly conserved, whereas most variation occurred in the middle region of ureB. An analysis of strains representing the 10 common types indicated that ureAB profiling was more discriminatory than ureC (HhaI) and ureC (MboI) profiling but marginally less discriminatory than combined ureC (HhaI-MboI) profiling (25). Although ureC (revised designation, glmM) is used for genotyping, its main role is now recognized to be related to cell wall synthesis and not to urease activity (4).

TABLE 2.

Signature bands for rapid identification of the main H. pylori ureAB profiles

Derived band size (bp)	Band presence in profile
	uAB1	uAB2	uAB10	uAB19	uAB3	uAB11	uAB4	uAB5	uAB6	uAB32
315	+a	+	+	+	+	+
457	+	+	+	+	+
772							+	+	+	+
444	+	+		+						+
827			+					+	+
295		+	+						+
678	+									+
Total no. of bands in profile	5	6	5	6	4	4	3	4	4	4

^a+, band was present in profile. Data do not include universally conserved and unique single bands. 

TABLE 3.

Analysis of genetic events within the ureAB operon of H. pylori

Change in uAB profile	Genetic eventa	Location of event (bp)
		Fragment(s)	Position (operon)
1→2	+	678	1599 (ureB)
1→19	+	678	1381 (ureB)
	+	678	1831 (ureB)
1→3	−	444, 678	1216 (ureB)
1→11	+	678	1831 (ureB)
	−	457, 444	772 (ureA)
2→10	−	315, 457	1216 (ureB)
10→6	−	772, 827	315 (ureA)
6→5	+	295	1831 (ureB)
5→4	−	827, 232	1599 (ureB)
4→32	−	63, 615	1831 (ureB)
	+	1059	1216 (ureB)
32→1	+	772	315 (ureA)

^aRestriction site gained (+) or lost (−). 

Our results confirm and extend the findings of previous studies showing considerable diversity in the ureAB region of H. pylori isolated from different individuals (2, 18). All strains produced functionally highly active urease, and mutations within the subunit genes were synonymous for the three sequenced strains. The effect of such variations on urease antigenicity is unknown but may be of practical significance when considering the effectiveness of urease-based vaccines in diverse and heterogeneous human populations.

A novel finding was that about half the strains were represented by just 10 ureAB RFLP profiles, which indicated a higher degree of conservation than expected. Furthermore, H. pylori with the same urease profile infected unrelated individuals, and in some cases, isolates were from widely separated parts of the world. Although it is generally believed that a large and very genetically diverse population of H. pylori circulates in the community, certain genomic markers, such as cagA and vacAs1, are widely distributed and partially conserved (8, 22), and it would appear that this is also true for certain ureAB polymorphisms. Further evidence of this conservation was apparent from published data on strains isolated in the United States showing profiles closely matching uAB3 and uAB5 (6). The availability of H. pylori urease gene sequences enabled precise sizes to be assigned to each RFLP profile and an exact genomic location to be identified. This novel development of RFLP analysis has considerable potential for studying allelic variation in other regions of the H. pylori genome, such as the highly polymorphic vacA locus (22).

In conclusion, we found that HaeIII RFLP analysis was an easy-to-perform, reproducible, and discriminatory means of documenting diversity within the H. pylori ureAB operon. Furthermore, differences were independent of variation in other loci associated with pathogenicity, such as cagA, vacA, 48-kDa HtrA, and 26-kDa antigen genes, although analysis of such loci with multiple restriction enzymes could be applied to further discriminate strains when necessary (2, 20). The scheme was developed mainly on U.K. isolates of H. pylori, but it appears to be equally applicable to isolates from other parts of the world and thus provides a practical general purpose genotyping framework to facilitate interlaboratory comparability.