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Mobley HLT, Mendz GL, Hazell SL, editors. Helicobacter pylori: Physiology and Genetics. Washington (DC): ASM Press; 2001.

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Helicobacter pylori: Physiology and Genetics.

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Chapter 33Heterogeneity and Subtyping

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Author Information

,1 ,2 ,2 and 3.

1 Helicobacter Reference Unit, Laboratory of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London, NW9 5HT, United Kingdom
2 Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada
3 Delft Diagnostic Laboratory, 2625, AD, Delft, The Netherlands

Introduction

Why Type Helicobacter pylori?

It is important to establish universal, reproducible, and discriminatory typing systems for most bacterial and viral pathogens. Likewise for H. pylori, which has a high prevalence in humans and a worldwide distribution, methods for typing strains are necessary for an understanding of its natural history, epidemiology, and clinical implications. Information on strain type is recognized to be of key importance in a number of areas, such as:

  • monitoring the effects of therapy and intervention procedures;
  • clues to the clonal and common ancestry of strains worldwide that might have relevance to design of vaccines;
  • associations of disease with a particular strain type;
  • animal model studies of infection;
  • relationships between type and drug resistance;
  • identification of strain factors that might contribute in assessing benefit of eradication in patients with non-ulcer dyspepsia;
  • investigation of routes of transmission;
  • identification of key reference strains.

Limitations of Present Phenotyping Methods

Various typing methods based on the differences of phenotypes among H. pylori isolates have been tested. They include the following:

Biotyping

Profiles of enzymatic activity (e.g., API Zym system) were suggested as a basis for biotyping of isolates but gave a low degree of discrimination (68, 97, 111).

Serotyping

H. pylori has both protein and common core lipopolysaccharides (LPS) group antigens and strain-specific side chain antigens. A provisional typing scheme with five serogroups was developed based on heat-labile (flagellar) antigens (74), but as most strains belonged to the same serogroup, the scheme lacked fine discrimination. The antigenicity of H. pylori LPS was also investigated (82), and sufficient diversity was found for potential use as the basis of an O-antigen (heat-stable) serotyping system comprising six types. However, the scheme was limited by low discrimination and cross-reactions with unabsorbed antisera.

Lewis blood group determinants

Lex and Ley determinants are present in about 80% of strains, and it was proposed they could form the basis of a discriminatory O-antigen serotyping scheme, using four specific monoclonal antibodies for detecting Lewis antigens in LPS (121). This scheme, however, is compromised by the frequent occurrence of phase variation.

Lectin typing

Lectins are sugar-binding proteins or glycoproteins of nonimmune (plant) origin and certain lectins were found to vary in their ability to bind to LPS and to agglutinate different strains. Recently, a typing scheme for H. pylori was developed comprising 16 possible lectin types (61).

On the basis of the results of these phenotyping schemes, H. pylori appears to be a surprisingly homogeneous species at the phenotypic level, with few features showing potential for typing purposes. Therefore, these phenotyping schemes are limited due to low discrimination and do not provide complete typeability for all isolates. Furthermore, the utility of such schemes is restricted, as appropriate typing sera and other reagents are not easily available. Another measure of the phenotype is provided by multilocus enzyme electrophoresis (MLEE), which analyzes the electrophoretic mobilities of the metabolic housekeeping enzymes and equates the different charges with alleles at the underlying genetic locus. Although such loci are highly polymorphic in H. pylori (50), MLEE has limited applications as a typing technique because it is laborious, subjective, and difficult to compare between laboratories.

Aims of This Chapter

The high level of genomic diversity among isolates of H. pylori infecting different individuals is widely documented and has provided the primary approach rather than the phenotyping for strain discrimination of the past decade. The purposes of this chapter are:

  1. To demonstrate the extent of genetic diversity observed in H. pylori and to illustrate the various approaches applied to investigate strain heterogeneity at the intragenic level (microdiversity) and throughout the genome (macrodiversity) as well as extragenomic or mobile elements such as plasmids and insertion sequences.
  2. To review genetic mechanisms causing diversity, and their possible effects on the discriminatory power and stability of typing schemes.
  3. To assess the potential of these approaches for strain typing in terms of discriminatory power, typeability, reproducibility, and general practical applicability.
  4. To identify aspects for future development.

Genotyping Based on Multiple Sites throughout the Genome and on Plasmids—Macrodiversity Analysis

Restriction Endonuclease Digest Analysis

Use of high-frequency cutting enzymes

DNA restriction digest pattern analysis was first applied to H. pylori in 1986 (71), and since then HaeIII and HindIII have been used widely for purposes that include comparisons of multiple isolate sets before and after treatment (14, 41, 78, 85, 89, 122), isolates from dental plaque and the stomach (119), isolates from different individuals in a family (12), and isolates from stomach corpus and antrum (19, 109). Most isolates from different individuals appear to have a unique DNA digest pattern. As the resultant digest patterns are highly complex with multiple bands, it is difficult to quantify similarities between strains and to define types based on groups of similar strains. A possible solution for subgrouping isolates is to use just the larger (10- to 20-kb) fragments to provide a signature pattern as applied to isolates compared before and after treatment (100).

Use of low-frequency cutting enzymes

An alternative approach is to compare isolates by pulsed field gel electrophoretic (PFGE) analyses of DNA digested with low-frequency cutting enzymes such as NotI and NruI (5, 113, 132, 133, 136) (Fig. 1A). These macro-restriction digests provide about four to nine well-resolved DNA fragments with high molecular weight (50 to 100 kb). PFGE has been applied in studies of strain heterogeneity (113) and multiple strain colonization (55, 132, 156), in checking strain identity (4), and in genetic map construction (136). Criteria for the general use of PFGE in bacterial typing have been proposed (140). The main disadvantage of PFGE analysis for H. pylori is the low typeability, as up to 40% of isolates may not be susceptible to analysis. Among possible reasons for this may be DNA modification/protection against digestion and DNA degradation during PFGE procedure. Nevertheless, in the case of strains that give good patterns with PFGE, this technique gives more information in terms of genome size and precise comparison of related strains (see comments in "Conclusions," below). PFGE can be applied effectively to several other species of Helicobacter, with excellent typeability and discrimination for H. mustelae (135), H. hepaticus (115), and H. pullorum (45).

Figure 1. Agarose gel-based DNA fingerprints used in genotyping strains of H.

Figure 1

Agarose gel-based DNA fingerprints used in genotyping strains of H. pylori. Panel A shows examples of PFGE patterns of NotI digests for various isolates including strain 60190 (lane 3, from left), NCTC11637 (lane 9), and NCTC11638 (lane 10). Panel B shows (more...)

Hybridization Analysis

Ribotyping

Ribosomal RNA gene patterns (ribopatterns) provide a simple yet highly discriminatory basis for strain identification (90). The pattern of bands reflects the presence and location of restriction sites within and flanking the two copies of the 16S and 23S rRNA genes. The overall complexity of the pattern is determined by both the restriction endonuclease used and the nature of the hybridization probe, which can be based either on the separate 16S and 23S rRNA gene sequences or on both sequences combined. The ribopatterns obtained with high-frequency cutting endonucleases such as HaeIII (4-bp recognition site) and HindIII or DraI (6-bp recognition site) are usually highly discriminatory and contain three to ten bands of 1 to 10 kb, although DNA from about 25% of H. pylori strains was not digested by HaeIII. In a more refined approach (28, 74), defined 16S rRNA gene probes and four restriction endonucleases (HaeIII, EcoRI, BstII, and BglII) were used to produce ribopatterns (see below). Since their first use in 1990 (85), ribopatterns have been applied widely to investigate H. pylori isolates from symptomatic and asymptomatic dyspeptic individuals. Other applications include characterization of H. pylori strains before and after treatment (41, 92, 94, 95, 110, 139), comparison of strains with different vacuolating cytotoxin activity (99), and comparison of isolates from different gastric sites (65, 98, 109). In general, duodenal and antral isolates from the same patient have similar ribotypes whereas strains from different individuals are distinct, except for some family members (12, 86). Cumulative data on ribotyping suggested that about half of the patients from the United Kingdom are each colonized by a single strain of H. pylori (91), while other patients appeared to be colonized by several strain types or subtypes.

The advantages of H. pylori ribotyping are its high reproducibility and the fragment size data that can be readily coded and used to construct databases (101). Overall, ribopatterns using an appropriate endonuclease and probe provide an excellent strain-specific fingerprint for small-scale studies of genomic variation in defined populations, such as sequential patient isolates. Laboratory procedures for conventional blot hybridization are time-consuming, but significant advances in automated ribotyping technology could have potential applications for H. pylori (57).

Other gene targets

H. pylori strain variation based on three urease genes (ureA, ureC, and ureD) was analyzed by genomic blot hybridization, and combined ureACD profiles were generated with HindIII (29). The most prevalent genotype was conserved among 33% of strains, and it was found that strains from patients with more severe forms of gastroduodenal disease were generally more homogeneous than strains carried by asymptomatic dyspeptics. In another approach, randomly cloned DNA probes were used to probe genomic blots for restriction site variation in and around homologous but undefined loci (73, 125). The degree of strain discrimination depended on the probe and the endonuclease used, reflecting the highly diverse organization of the H. pylori genome. In a subsequent study, another cloned H. pylori-specific chromosomal DNA fragment was evaluated by blot hybridization of HaeIII-digested chromosomal DNA and the result was compared with that using a 16S ribotyping probe (28). Short oligonucleotide probes containing repetitive sequences also can be used for typing (79). In conclusion, these studies indicated that gene-specific hybridization probes, including those for rrn genes, are highly discriminatory and provide a valuable basis for strain typing.

PCR-Based Techniques Using Arbitrary Sequences

Repetitive extragenic palindromic sequences

Repetitive extragenic palindromic (REP) DNA sequences have been identified in many bacterial species and have been detected in the H. pylori genome by PCR (48) and sequencing (6, 142). Repetitive DNA sequence-based PCR (REP-PCR), which refers to the general method utilizing oligonucleotide primers of about 20 bp that match interspersed repetitive DNA sequences, was used to generate H. pylori strain-specific DNA fingerprints, and cluster analysis grouped strains according to duodenal or gastric ulcer associations (48). The presence of specific virulence determinants could not be demonstrated by REP-PCR without the use of blot hybridization combined with DNA sequence analysis (70). REP-PCR was also applied to fingerprint single colonies from the antrum, corpus, and duodenum of patients with gastric cancer (81); to compare strains among family members (80); and to investigate the emergence of antibiotic resistance (32). REP-PCR offered greater typeability and discrimination than PFGE (156). A modification of this technique using end-labeled fragments combined with numerical analysis revealed no clustering of disease-specific strains (154).

Arbitrary primer PCR profiling

Fingerprinting using random amplified polymorphic DNA (RAPD) or arbitrary primer (AP-PCR) provides a high degree of discrimination between clinical isolates of H. pylori (2). The method uses oligonucleotides of arbitrarily chosen sequences to prime DNA synthesis from pairs of sites with complete or partial matches, resulting in strain-specific arrays of DNA products. Several 10-mer primers with high G + C content yield strain-specific arrays of up to 15 prominent bands. RAPD was applied to analyze populations of H. pylori in antral biopsies from individual patients (79), to measure long-term colonization (up to 4 years) after antibiotic treatment (138), to study reinfection or new infection (163) and coinfection in children (34), and to identify different cultures of NCTC11637 (4). These studies demonstrated the speed and high discriminatory power of RAPD typing for H. pylori, although no generally recognizable types were identified.

Amplified fragment length polymorphism

Amplified fragment length polymorphism (AFLP) analysis is a new technique in which adapter molecules are ligated to restriction enzyme fragments and subsequently used as target sites for PCR primers. AFLP is applicable to fingerprinting a wide variety of microbial species, and the single-restriction endonuclease approach (144) was modified to characterize isolates of H. pylori. In a study of 46 isolates from 28 dyspeptic patients, AFLP profiles derived from HindIII fragments differentiated strains from unrelated individuals and confirmed the common origin of strains in some family members (Fig. 1B) (47). AFLP analysis offers the advantages of speed and reproducibility and also provides a means of examining DNA segments distributed over the entire genome of a microorganism. This is an advantage over other methods monitoring restriction site changes within single loci. However, similar to AP-PCR and RAPD genotyping, AFLP produces complex fragment profiles that are difficult to interpret, and thus is inconvenient for defining common genotypes.

Plasmids and Insertion Sequences

Plasmids have been detected in H. pylori and were present in up to 40% of isolates in some studies (92, 93, 96, 98, 108, 122, 125, 141). Strains carrying plasmids can be grouped both on the basis of the number of plasmids present and on their sizes, and such details may be a useful adjunct for strain characterization (125). For instance, a 5,846-bp plasmid was characterized in the Sydney strain SS1, and common regions between plasmids and the chromosome were identified as indicative of plasmid-mediated recombination (30). Plasmid profiling provides low typeability and low strain discrimination so it has only limited potential as a typing method for H. pylori.

Various insertion sequences (IS elements) are also present in the H. pylori genome. For instance, IS605 was found when sequencing virulence-associated genes in reference strain NCTC11638 (20). Five complete and eight partial copies of IS605 were present in the genome of strain 26695 (142) whereas no complete copy and five partial copies were present in the J99 genome (6). Strain 26695 also contains two full copies and two partials of a related insertion element, IS606. The J99 genome contained one complete and several partial IS606 sequences. Frequencies of IS605-carriage of up to 31% have been reported on the basis of PCR assays, and IS presence appears to be independent of geographical origin of the isolate (58, 123). In general, the relatively low frequency of occurrence of IS elements in the H. pylori genome appears to limit the usefulness of IS typing.

Genotyping Based on Known Loci—Microdiversity Analysis

cagA, the Cytotoxin-Associated Gene

The cytotoxin-associated gene (cagA) is present in approximately 50 to 70% of H. pylori strains (21, 24, 123, 143) and is considered a marker for the presence of a genomic pathogenicity island (cag PAI) of about 40 kb (3, 20, 22). Presence of this cag PAI is associated with more severe clinical outcomes in Western populations (13, 53, 67, 106, 112). In contrast, in Asian populations, virtually all H. pylori strains are cagA+ and the association between cagA positivity and disease is much weaker or not present (49, 88, 105). Also, a mixture of cagA+ and cagA strains may be present in a single patient (36, 38, 123, 147).

Most cagA+ strains contain a contiguous cag PAI (123) and the gene order is relatively well conserved (3). In some strains, however, the island is split into a right segment (cagI) and a left segment (cagII) by the insertion sequence IS605 (20). Some strains only contain part of the cag PAI (76). CagA, which induces growth changes in human epithelial cells (118), constitutes a major antigen of H. pylori (143) and elicits specific anti-CagA antibodies. Several studies assessed the sequence heterogeneity of cagA, and considerable variation was found by restriction fragment length polymorphism (RFLP) (102). Initial sequencing studies, comparing a limited number of H. pylori strains, indicated that the 5′ part of cagA is more conserved than the middle and 3′ end of the gene, which contains repeated sequences (22, 143). These repeats may be associated with the levels of anti-CagA antibody, the degree of atrophy, enhanced histological injury, and reduced survival in acidic conditions (164, 166).

The diversity of the 5′ end of the gene was studied in more detail by PCR and direct sequence analysis in strains from various geographic origins (146, 152). Phylogenetic analysis revealed the existence of at least two distinct types of cagA. One variant (cagA1) was found exclusively in strains from Europe, the United States, and Australia, whereas a novel variant (cagA2) was found in strains from East Asia. The greatest diversity between cagA1 and cagA2 was found in the first 20 amino acids predicted by the cagA open reading frame (ORF), in which several conserved insertions or deletions were observed.

cagA can be detected by PCR, but due to the sequence heterogeneity, the efficacy of primers should be evaluated for different patient populations. The presence of the "empty site," indicating absence of the cag PAI, also can be determined by PCR (123).

vacA, Encoding the Vacuolating Cytotoxin

vacA encodes a toxin that is secreted by H. pylori and causes vacuoles in epithelial cells (23, 25, 72). vacA is present in all H. pylori strains, and two parts of the gene show particular sequence heterogeneity (10). The s-region (encoding the signal peptide), located at the 5′ end of the gene, occurs as an s1 or s2 type. Within type s1, several subtypes (s1a, s1b, and s1c) can be distinguished (151). Within the m-region (middle), m1 or m2 types exist, and within m2, subtypes m2a and m2b can be recognized. The mosaic combination of these distinct s- and m-region allelic types correlates with the specific activity of the cytotoxin and is associated with the pathogenicity of the bacterium (10, 52, 153, 159).

vacA genotypes can be distinguished by different methods, such as type-specific PCR (10, 11), PCR-RFLP (Fig. 1C) (31, 54, 103), and the PCR-line probe assay (LiPA) (151), permitting direct analysis of gastric biopsy specimens (Fig. 2). As for detection of cagA, the efficacy of vacA-specific primer sets should be carefully evaluated in different patient populations (149).

Figure 2. Genotyping of H.

Figure 2

Genotyping of H. pylori isolates using reverse hybridization. Panel A shows the principle of the reverse hybridization line probe assay (LiPA). Specific oligonucleotide probes are tailed and immobilized onto membrane strips in parallel lines. Biotinylated (more...)

vacA and cagA genotypes of H. pylori have a particular geographic distribution (18, 149). H. pylori strains from East Asian patients often contain subtype vacA s1c, whereas this type is very rare in other parts of the world. Non-Asian patients carrying s1c strains were all born in East Asia or have Asian ancestors. In Europe, a distribution gradient was observed. Most strains from northern, central, and eastern Europe are of subtype s1a. In France and Italy, s1a and s1b were present in equal frequencies, whereas in Portugal and Spain, most strains were of subtype s1b. Also, most South American strains were of subtype s1b. There also is a close association between the presence of cagA and vacA type s1 (10). Most cagA2 strains contained vacA subtype s1c, and cagA1 was more frequently observed in vacA m1 strains than cagA2. These results show the epidemiological relationship between cagA and vacA at the subtype level and indicate the existence of distinct H. pylori lineages that are not uniformly distributed over the globe. These findings strongly indicate comigration and coevolution of H. pylori and humans since ancient times.

Urease-Encoding Genes

The H. pylori urease is composed of six copies each of two subunits, UreA and UreB, and is the major protein synthesized by H. pylori. Since production of urease is obligatory for the survival of H. pylori, the urease operon, comprising ureABEFGHI, has been studied extensively and is a popular target for sensitive molecular detection of infection (16, 75, 161). Polymorphisms in the urease genes have been investigated by PCR-RFLP, RAPD, and sequencing to compare strains from different patient groups (29, 40, 63, 84, 103, 104, 114, 134), but no relation between any particular genotype and disease was found. Sequence variation in the urease genes also permitted identification of specific strains during treatment follow-up (126) and comparison of strains from various sites of the stomach in individual patients (42). PCR-RFLP analysis of the ureAB genes permitted comparison of H. pylori strains from geographically diverse regions, from different family members, and pre- and post-treatment strains (94, 104).

flaA and flaB, the Genes for Flagellins

Flagellar motility of H. pylori is important for effective colonization (35). The major and minor flagellins are encoded by flaA and flaB, respectively (130). To study the population structure of H. pylori, sequences of flaA and flaB have been analyzed for different strains and revealed extensive heterogeneity (131). PCR-RFLP analysis showed extensive sequence variation of flaA, allowing discrimination between individual strains. However, the RFLP profiles could not be associated with strain phenotype or clinical symptoms of the patient (60). As for vacA and cagA subtypes, flaA alleles showed a particular geographical distribution, confirming that H. pylori variants are not uniformly distributed around the world (39).

iceA, Induced upon Contact with Epithelium

Prior to genomic sequencing, the gene iceA (induced by contact with epithelium) was characterized; it exists as two distinct types, iceA1 and iceA2 (37, 107). iceA1 possesses significant homology to a type II restriction endonuclease from Neisseria lactamica. iceA2 has a particular repeat structure, predicting several distinct, highly conserved protein motifs, and is preceded by a variable number of 8-bp repeats, which permit discrimination between individual strains. The function of IceA2 remains unknown (37). The expression of iceA1 is upregulated upon contact between H. pylori and human epithelial cells, and the presence of this allele is associated with peptic ulcer disease in some populations (107, 153) but not in others (165).

babA, Associated with Binding to Blood-Group Antigens

Genes encoding outer membrane proteins that mediate binding of H. pylori to the blood group antigen Lewis B on human host cell surface have been designated bab (62). babA exists in two distinct genotypes, babA1 and babA2. babA1 comprises an almost complete ORF but lacks the necessary initiation codon and is therefore not translated. In contrast, babA2 contains a 10-bp insert at its 5′ end that includes an in-frame ATG start codon. This permits expression of the babA2 ORF, which correlates with bacterial adhesion. Together with vacA s1 and cagA, babA2 is strongly associated with peptic ulcer disease and adenocarcinoma (43). Apart from the 10-bp insert, the sequences of babA1 and babA2 are highly similar. Moreover, the 5′ end of another outer membrane protein gene (designated OMP9) is highly similar to babA. Therefore, specific typing of the babA genes is not easy and requires carefully designed PCR primers.

Mutations Associated with Macrolide Resistance Used for Typing Strains

Macrolides can bind to specific regions of the 23S rRNA and inhibit ribosomal function. Specific mutations in the 23S rRNA peptidyltransferase domain (domain V) change its conformation. Consequently, binding of macrolides is prevented, resulting in antibiotic resistance. Point mutations, associated with macrolide resistance, have been found at positions 2115, 2141, 2142, and 2143 of either of the two copies of the H. pylori 23S rRNA gene (26, 27, 59, 87, 137, 155). The A2142G and A2143G mutations are most common among clarithromycin-resistant strains. Macrolide resistance can be easily determined by sequencing, ligase chain reaction (LCR) (127, 128), RFLP (27), specific probe hybridization in microtiter plates (87), real-time PCR with the LightCycler (46), and reverse hybridization LiPA (148) (Fig. 2). These mutations also can be detected directly in gastric biopsies, omitting the need for time-consuming culture of the bacterium. Since the prevalence of macrolide-resistant strains appears to be increasing in various parts of the world, these molecular methods will become increasingly important.

Multilocus Sequence Typing

Although sequence analysis of complete genomes is the most accurate method to distinguish genetic variants, this approach is neither feasible nor necessary. Sequencing of a representative selection of fragments distributed over the bacterial genome also permits detailed genetic analysis of individual strains. This method, designated as multilocus sequence typing (MLST), has been successfully applied to Neisseria meningitidis and H. pylori, and several other bacterial species are currently under investigation (1, 77). MLST has a very high resolution but requires high-volume sequencing, as well as detailed phylogenetic analysis of multiple sequences.

Mechanisms of Genetic Variation in Relation to Genotyping

Mutation

Mutations arise from errors in copying normal template DNA and misreplication of DNA damage. In the published H. pylori genome sequences (6, 142), some gene homologs functioning in DNA damage reversal (e.g., Escherichia coli homologs mutM, nfo, vsr, oxyR, ada, and ogt) are not present, suggesting that H. pylori is not able to directly remove DNA lesions as efficiently as E. coli. Also, H. pylori lacks an E. coli mutHLS-like postreplicative mismatch repair system (157). Through binding to the mismatched base pairs, the mutHLS system has two functions: preventing occurrence of mutations from replication errors and inhibiting homologous recombination between diverged DNA sequences (83). Absence of the mutHLS system in H. pylori may predict an increased mutation rate and lack of genetic barrier for interstrain recombination (see below).

Frequencies of spontaneous mutations in most gene loci can be as low as 10−10 per base pair per generation. Particular nucleotide sequence contexts could be more easily mutated than others. The one type of mutation hotspot that can easily be identified is the simple sequence repeat (SSR) (145). During replication, DNA polymerase slips easily at SSRs, leading to mispairing between the template and the newly synthesized strand (slipped-strand mispairing). By this mechanism, addition or deletion of one or a few repeat units can occur at a much higher frequency than the normal mutation rate. Such frameshift mutations can lead to the on-off switch of the target gene. A number of H. pylori genes have been identified that contain SSR, including those encoding outer membrane proteins, enzymes for LPS biosynthesis, and DNA restriction-modification systems (6, 116, 142). Recently, experimental evidence was presented for the molecular basis of the phase variation of Lewis antigen expression mediated by slipped-strand mispairing in fucosyltransferase genes (8, 158). Mutation hotspots other than SSR have yet to be identified in H. pylori.

DNA Transformation

The majority of H. pylori strains are naturally competent for DNA transformation (160). Natural transformation is a complex process involving DNA uptake followed by recombination (see chapter 22). Evidence has been presented that a gene cluster consisting of four genes (orf2, comB1, comB2, and comB3) (56) and the dprA gene (7, 124) are involved in natural competence and DNA uptake.

In several well-studied naturally transformable bacteria species, specific DNA sequences of approximately 10 bp that are abundant throughout the genome are required for DNA uptake, which ensures that the substrate for recombination is derived from related bacteria (33). By searching for the frequent short sequences in the H. pylori genome, Saunders et al. (117) concluded that H. pylori does not possess such an uptake sequence. Thus, H. pylori might be expected to have an increased likelihood of taking up DNA from unrelated species when it is encountered. It is also possible that very small pieces of H. pylori DNA (from the cells of the same strain or different strains) are taken up into the cell for the use of DNA recombinational repair (see below).

DNA Recombination

After uptake, the DNA may become the substrate for homologous recombination. Genetic recombination in H. pylori is so frequent that it randomizes the DNA sequences and generates linkage equilibrium (a so-called panmictic population structure) (1, 131). Kersulyte et al. (64) provided in vivo evidence of interstrain recombination of H. pylori. Cocolonization of multiple strains in one human host provides the opportunity for interstrain recombination. Approximately 10% of individuals in the developed world appear to be colonized with more than one strain (120, 138, 153), whereas in developing countries, up to 34% of individuals tested have been found to be colonized with multiple strains (15, 150).

E. coli has two recombination pathways: the primary RecBC pathway mainly serves the needs of conjugational recombination, and the secondary RecF pathway functions mainly in recombinational repair when the primary pathway is inactive (69). According to the complete genome sequences (6, 142), the RecBC pathway is absent in H. pylori. The genes coding for components of the RecF pathway (recJ, recN, recR, and recG) are present, in addition to those for the essential components common to both pathways, recA and ruvABC. It appears that the RecF pathway is the only pathway in H. pylori, and its main function is for recombinational repair. Natural competence for DNA uptake, lack of a DNA uptake sequence, and lack of a mutHLS-like system suggest that H. pylori has a highly efficient mechanism of recombinational repair.

Interspecies Gene Transfer

Some H. pylori genes share significant sequence similarity to those of gram-positive bacteria, archae, and eukaryotes. For example, the entire biosynthetic pathway for cytochrome c biogenesis in H. pylori is characteristic of that found in gram-positive bacteria (51). The complete genome sequences (6, 142) revealed that several DNA segments, including the cag PAI, have a significantly different G + C content compared to the mean content of 39% for the entire genome, suggesting that they may have originated from other species. Some of these regions contain IS605, implicating a role of this insertion sequence in lateral transfer of these DNA segments into H. pylori.

Horizontal transfer of genes from other species may have occurred in the evolution of the species. Because of the lack of homologous target gene sequence in the recipient, interspecies gene transfer is usually mediated by plasmids, transposons, or IS elements or by an illegitimate recombination event. These events occur rarely compared to normal homologous recombination. In addition, presence of many modification-restriction systems in H. pylori could be an additional inhibiting factor for interspecies gene transfer.

Relevance to Genotyping

Different genetic events occur at different frequencies, which merits consideration when selecting appropriate genotyping schemes. Mutation and recombination are two major driving forces generating DNA polymorphism. Mutations can occur de novo or can accumulate by vertical transmission from previous generations. By recombination, DNA polymorphism can be generated much more rapidly than by mutation alone.

Many genetic markers of H. pylori investigated are rather stable (in vitro or in vivo) in a short time period (94, 103, 113). Genetic changes were observed in some other studies. For example, van der Ende et al. (147) observed genetically related H. pylori strains among members of the same family, with variation between and within individuals. Recently, Kuipers et al. (66) showed that over a period of 7 to 10 years, some strains changed their RAPD-PCR and AFLP patterns, although the particular loci such as cagA and vacA remain unchanged, suggesting that mutations have developed over the course of prolonged colonization. Because of the high frequency of mutation in fucosyltransferase genes, the phenotypic changes of Lewis antigen expression are frequently observed (9, 44, 66, 162).

Genetic drift could be facilitated during transmission when H. pylori cells may be exposed to environmental stresses. Under certain conditions such as acidic pH and oxidative stress, cells suffer more DNA damage, which facilitates both mutation and recombination. In addition, the constant load of other bacteria passing through the stomach as well as putative host factors might have an impact on the genetic stability of the resident H. pylori.

Besides the molecular genetic events mentioned above, natural selection has an important role in generating genetic diversity. The majority of mutations are synonymous due to the selective pressure. Therefore, genotyping methods monitoring only DNA base substitutions may lead to overestimation of the extent of diversity (6). By natural selection, most mutations are removed from the gene pool. rRNA genes and many housekeeping genes are under higher selective pressure than other genes. Genotyping based on these conserved loci gives more information on evolutionary relatedness of H. pylori isolates. Variation in some gene loci could be tolerated for bacterial survival, and certain subtypes could be more adaptive than others to a particular niche. Particular genotypes of certain loci such as cagA, vacA, babA, and iceA may be related to particular disease states and thus have clinical implications.

Conclusions

Guidelines for Genotyping: Matching Aims with Methods

Genomic diversity within H. pylori is now well established and a plethora of molecular typing techniques is available, many of which have been applied successfully to distinguish between isolates. However, no single molecular method has yet emerged as an internationally accepted basis for typing or subtyping purposes. Although consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems have been proposed (129), they are aimed mainly at phenotypic methods applicable in the characterization of isolates implicated in community or hospital outbreaks.

For H. pylori, the situation is perhaps unique as no reliable phenotyping methods are available for comparison, and outbreaks of infection, which usually provide an important basis for validating typing methods, are not a feature of the epidemiology of the species. The earliest typing methods used for H. pylori, such as restriction digest analysis (including PFGE), ribotyping, and other forms of blot hybridization, have been now largely superceded by PCR-based methods such as RAPD, REP, and RFLP analysis. This trend was highlighted in a recent analysis of performance criteria for H. pylori typing methods, in which the superiority of the PCR methods was demonstrated, including better typeability, higher discrimination power, and better general ease of use (17).

While there are a number of important reasons to characterize individual strains of H. pylori, the typing method employed is largely dictated by the specific aim of the investigation, and there is no single method that can satisfy all requirements. Some guidelines are provided in Table 1 for various typing methods and the purposes for which they might be most appropriate. They are grouped in three general categories, i.e., markers for potentially enhanced pathogenicity, precise identification of strain differences, and global population structure analysis. Some comments on the relative advantages and disadvantages of the different methods are provided.

Table 1. Guidelines to different H. pylori genotyping methods and their general applications.

Table 1

Guidelines to different H. pylori genotyping methods and their general applications.

Future Needs

The enormous expansion in detailed information about the fine genetic structure of H. pylori, promoted by the availability of two complete genome sequences, continues to focus interest on strain-specific attributes, and hence a wider recognition of the importance of precise methods for strain identification. The present evidence is that strain genotypes appear to be relatively stable, at least under in vitro conditions. However, in view of the in vivo plasticity of the H. pylori genome, it will be essential to monitor how typing markers are affected by genetic drift. Aspects that could merit development in the future include direct typing from clinical specimens without the need for culture, development of global standardized data sets of genomic fingerprints for different PCR-based methods, and development of sequence-based typing using standard sets of loci in housekeeping as well as pathogenicity marker genes.

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