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Susceptibility testing was performed at seven Canadian microbiology laboratories and the Helicobacter Reference Laboratory, Halifax, Nova Scotia, Canada, to assess susceptibility testing proficiency and the reproducibility of the results for clarithromycin and metronidazole and to compare the Epsilometer test (E test) method to the agar dilution reference method. Control strain Helicobacter pylori ATCC 43504 (American Type Culture Collection) and 13 clinical isolates (plus duplicates of four of these strains including ATCC 43504) were tested blindly. The National Committee for Clinical Laboratory Standards (NCCLS) guidelines for agar dilution testing were followed, and the same suspension of organisms was used for agar dilution and E test. Antimicrobials and E test strips were provided to the investigators. Methods were provided on a website (www.Helicobactercanada.org). Each center reported MICs within the stated range for strain ATCC 43504. Compared to the average MICs, interlaboratory agreements within 2 log(2) dilutions were 90% (range, 69 to 100%) for clarithromycin by agar dilution, with seven very major errors [VMEs], and 85% (range, 65 to 100%) by E test, with three VMEs. Interlaboratory agreements within 2 log(2) dilutions were 83% (range, 50 to 100%) for metronidazole by agar dilution, with six VMEs and eight major errors (MEs), and 75% (range, 50 to 94%) by E test, with four VMEs and four MEs. At lower and higher concentrations of antibiotic, E test MICs were slightly different from agar dilution MICs, but these differences did not result in errors. When a standardized protocol based on NCCLS guidelines was used, most participants in this study correctly identified clarithromycin- and metronidazole-susceptible and -resistant strains of H. pylori 93% of the time by either the agar dilution or E test method, and the numbers of errors were relatively equivalent by both methods.

Although Helicobacter pylori express Lewis antigens as a component in the lipopolysaccharide, their role in the infectious process is not well understood. Lewis antigen expression with growth phase was investigated, as well as the distribution of Lewis antigens among isolates from asymptomatic and symptomatic individuals. Lewis antigens are expressed by H. pylori in a growth phase-dependent manner, with the greatest expression occurring in the logarithmic phase of growth. As growth proceeds, an increasing amount of Lewis antigens are shed into the culture supernatant. Lewis antigen expression among H. pylori isolates from asymptomatic individuals is characterized by an absence of type I Lewis antigens, a decrease in Le(x) expression, and an increase in nontypeable H. pylori, as compared with that among H. pylori isolates from symptomatic patients. The data support a role for Lewis antigens in the pathogenesis associated with symptomatic H. pylori infection in colonized individuals.

Helicobacter pylori are found attached to mucous cells of the human stomach or under the mucous layer. Models mimicking the in vivo situation may be more suitable for H. pylori MIC determinations than traditional agar dilution methods. Megraud et al. (Antimicrobial Agents and Chemotherapy 1991, 35, 869-72) developed a model for measuring the susceptibility of attached and free-floating H. pylori. We have modified this model so that free-floating and attached H. pylori are treated in a more similar manner, before and after incubation with antibiotic, and performed additional controls to ensure H. pylori and tissue culture cells are not detrimentally affected and maintain their viability during the course of the experiment. We found only 10% of plate-grown H. pylori were competent for attachment to HEp-2 cells; however, all progeny of attached bacteria remained adherent. Killing curves were performed using 0, 0.001, 0.01, 0.1 and 1 mg/L amoxycillin, and 0, 0.0025, 0.0075 and 0.01 mg/L clarithromycin. H. pylori divided at concentrations

In this study, we systematically examined in vitro frequencies and spectra of the spontaneous mutations in Helicobacter pylori that confer resistance to clarithromycin (Cla(r)), metronidazole (Mtz(r)), amoxicillin (Amx(r)), ciprofloxacin (Cip(r)), and rifampin (Rif(r)). The mutation rate of Rif(r) or Cip(r) determined in a fluctuation assay is 1 x 10(-8) to 2 x 10(-8) per cell per division. In contrast, the mutation rates of Cla(r), Mtz(r), and Amx(r) are much lower (<10(-9)). However, Mtz(r) mutants could be readily selected in vitro by using the serial passage method, suggesting that the mutagenic effect and selective effect of a sublethal dose of metronidazole contribute to the rapid development of Mtz(r). Analysis of spontaneous Rif(r), Cla(r), and Cip(r) mutants confirmed previous results indicating that mutations within the rpoB gene, the 23S rRNA gene, and the gyrA gene, respectively, are responsible; also, several new mutant alleles were identified. Mtz(r) mutants resulted most frequently, but not always, from mutations in the rdxA gene. DNA fragments containing each mutant allele could readily transform susceptible H. pylori strains to resistance, confirming that each mutant allele is responsible for the resistance phenotype.

Helicobacter pylori is a widespread Gram-negative bacterium responsible for the onset of various gastric pathologies and cancers in humans. A familiar trait of H. pylori is the production of cell-surface lipopolysaccharides (LPSs; O-chain --> core --> lipid A) with O-chain structures analogous to some mammalian histo-blood-group antigens, those being the Lewis determinants (Lea, Leb, Lex, sialyl Lex, Ley) and blood groups A and linear B. Some of these LPS antigens have been implicated as autoimmune, adhesion, and colonization components of H. pylori pathogenic mechanisms. This article describes the chemical structures of LPSs from H. pylori isolated from subjects with no overt signs of disease. Experimental data from chemical- and spectroscopic-based studies unanimously showed that these H. pylori manufactured extended heptoglycans composed of 2- and 3-linked D-glycero-alpha-D-manno-heptopyranose units and did not express any blood-group O-antigen chains. The fact that another H. pylori isolate with a similar LPS structure was shown to be capable of colonizing mice indicates that H. pylori histo-blood-group structures are not an absolute prerequisite for colonization in the murine model also. The absence of O-chains with histo-blood groups may cause H. pylori to become inept in exciting an immune response. Additionally, the presence of elongated heptoglycans may impede exposure of disease-causing outer-membrane antigens. These factors may render such H. pylori incapable of creating exogenous contacts essential for pathogenesis of severe gastroduodenal diseases and suggest that histo-blood groups in the LPS may indeed play a role in inducing a more severe H. pylori pathology.

Resistance of Helicobacter pylori to antibiotics ranges from 3% to 10% and may exceed these levels in some countries. The pathophysiology of clarithromycin resistance is reviewed, including the mode of action by which the antibiotic inhibits protein synthesis and the mechanism of resistance, which involves a mutation at position 2142 or 2143 in the V loop domain of the 23SrRNA genes. Mutations of A2142G confer a higher minimum inhibitory concentration than mutations of A2143G. The former demonstrate cross-resistance to macrolide, lincosamide and streptogramin antibiotics, whereas the latter are susceptible to streptogramin B. In vitro mutagenesis combined with natural transformation were used to create several types of clarithromycin-resistant mutants. H pylori strains with A2142G and A2143G mutations had a higher growth rate than those with A2142C, A2143 or A2142T mutations. Data from this study indicate why clarithromycin-resistant clinical isolates of H pylori are more likely to have A2142G or A2143G mutations and only occassionally A2142C mutations.

The identification of Helicobacter pylori isolates that expresses exclusively type I Lewis antigens is necessary to determine the biosynthetic pathway of these antigens. Fast-atom bombardment MS provides evidence that the H. pylori isolate UA1111 expresses predominantly Leb, with H type I and Lea in lesser amounts. Cloning and expression of the H. pylori fucosyltransferases (FucTs) allow comparisons with previously identified H. pylori enzymes and determination of the enzyme specificities. Although all FucTs, one alpha(1,2) FucT and two alpha(1,3/4) FucTs, appear to be functional in this isolate, their activities are lower and enzyme specificities are different to other H. pylori FucTs previously characterized. Studies of the cloned enzyme activities and mutational analysis indicate that Lea acts as the substrate for the synthesis of Leb. This is different from the human Leb biosynthetic pathway, but analogous to the biosynthetic pathway utilized by H. pylori for the production of Ley.

The lipopolysaccharides (LPS) of most Helicobacter pylori strains contain complex carbohydrates known as Lewis antigens that are structurally related to the human blood group antigens. Investigations on the genetic determinants involved in the biosynthesis of Lewis antigens have led to the identification of the fucosyltransferases of H. pylori, which have substrate specificities distinct from the mammalian fucosyltransferases. Compared with its human host, H. pylori utilizes a different pathway to synthesize the difucosylated Lewis antigens, Lewis y. and Lewis b. Unique features in the H. pylori fucosyltransferase genes, including homopolymeric tracts mediating slipped-strand mispairing and the elements regulating translational frameshifting, enable H. pylori to produce variable LPS epitopes on its surface. These new findings have provided us with a basis to further examine the roles of molecular mimicry and phase variation of H. pylori Lewis antigen expression in both persistent infection and pathogenesis of this important human gastric pathogen.

Background and Aims: A mouse model was established to compare colonization by the H. pylori Sydney strain SS1 with several clinical isolates expressing different Lewis antigens on their surface. In addition, both humoral and cell mediated immune responses were determined for different H. pylori strains., Methods: Mice were inoculated intragastrically separately with the Sydney strain as well as with five clinical isolates of H. pylori expressing different Lewis (Le) antigen phenotypes. Colonization of the mouse stomach by the bacteria was monitored from two to fourteen weeks post inoculation by four independent methods namely, urease, PCR (using CagA primers), bacterial culture and histology. Antibody titers and cellular immune responses were monitored by ELISA and antigen stimulation test respectively., Results: Different degrees of colonization were observed in C57, CD1 and Balb/c mice inoculated with H. pylori strain SS1 (Le(x), Le(y)) and clinical isolates UA948 (Le(a), Le(x)), UA861 (alpha-glucosyl polyLacNAc), UA1258 (Le(y)), UA802 (Le(y)) and UA1264 (no Le antigen) starting from week two post inoculation. All three mice strains mounted high immune responses against different H. pylori antigens. Treatment of mice with vancomycin prior to inoculation has no effect either on colonization of the stomach or the immune response of the mice. Histological evaluation established colonization after 10 weeks post inoculation but not gastritis., Conclusions: Stomach of mice can be colonized consistently, with H. pylori strain SS1, and colonization was also achieved with all clinical isolates that were not mouse adapted. These strains could be detected more consistently by PCR in the early stages, then by culture only after 8 - 10 weeks. In our study, Lewis(x) expressing bacterial strain (UA948) failed to colonize Balb/c mice, whereas the Le(y) expressing strain (UA1258) did not colonize C57/BL6 mice.

The expression of Lewis antigens by the gastric pathogen Helicobacter pylori in serial biopsy isolates was investigated to assess antigen distribution and stability. A total of 26 asymptomatic subjects were given various doses of 3' sialyllactose for up to 56 days. Gastric biopsies were performed during the dosing period, as well as 30 days after dosing, which provided 127 H. pylori isolates that were examined by use of ELISA and immunoblot. A large proportion of subjects (14/26) yielded sequential H. pylori isolates, which appeared genetically identical but had variable Lewis antigen expression. The proportion of subjects with H. pylori isolates not expressing any Lewis antigens was greater than that previously reported (10/26). Thus, the expression of the Lewis antigens by H. pylori does not appear to be a requirement for colonization, whereas the antigen expression after human infection is more variable than the previously reported rate observed with in vitro cultures.

The gastric pathogen Helicobacter pylori can express the histo blood group antigens, which are on the surface of many human cells. Most H. pylori strains express the type II carbohydrates, Lewis X and Y, whereas a small population express the type I carbohydrates, Lewis A and B. The expression of Lewis A and Lewis X, as in the case of H. pylori strain UA948, requires the addition of fucose in alpha1,4 and alpha1,3 linkages to type I or type II carbohydrate backbones, respectively. This work describes the cloning and characterization of a single H. pylori fucosyltransferase (FucT) enzyme, which has the ability to transfer fucose to both of the aforementioned linkages in a manner similar to the human fucosyltransferase V (Fuc-TV). Two homologous copies of the fucT gene have been identified in each of the genomes sequenced. The characteristic adenosine and cytosine tracts in the amino terminus and repeated regions in the carboxyl terminus are present in the DNA encoding the two UA948fucT genes, but these genes also contain differences when compared with previously identified H. pylori fucTs. The UA948fucTa gene encodes an approximately 52-kDa protein containing 475 amino acids, whereas UA948fucTb does not encode a full-length FucT protein. In vitro, UA948FucTa appears to add fucose with a greater than 5-fold preference for type II chains but still retains significant activity using type I acceptors. The addition of the fucose to the type II carbohydrate acceptors, by UA948FucTa, does not appear to be affected by fucosylation at other sites on the carbohydrate acceptor, but the rate of fucose transfer is affected by terminal fucosylation of type I acceptors. Through mutational analysis we demonstrate that only FucTa is active in this H. pylori isolate and that inactivation of this enzyme eliminates expression of all Lewis antigens.

This study describes the molecular makeup of the cell-wall lipopolysaccharides (LPSs) (O-chain polysaccharide-->core oligosaccharide-->lipid A) from five Helicobacter pylori strains: H. pylori 26695 and J99, the complete genome sequences of which have been published, the established mouse model Sydney strain (SS1), and the symptomatic strains P466 and UA915. All chemical and serological experiments were performed on the intact LPSs. H. pylori 26695 and SS1 possessed either a low-Mr semi-rough-form LPS carrying mostly a single Ley type-2 blood-group determinant in the O-chain region covalently attached to the core oligosaccharide or a high-Mr smooth-form LPS, as did strain J99, with an elongated partially fucosylated type-2 N-acetyllactosamine (polyLacNAc) O-chain polymer, terminated mainly by a Lex blood-group determinant, connected to the core oligosaccharide. In the midst of semi-rough-form LPS glycoforms, H. pylori 26695 and SS1 also expressed in the O-chain region a difucosylated antigen, alpha-L-Fucp(1-3)-alpha-L-Fucp(1-4)-beta-D-GlcpNAc, and the cancer-cell-related type-1 or type-2 linear B-blood-group antigen, alpha-D-Galp(1-3)-beta-D-Galp(1-3 or 4)-beta-D-GlcpNAc. The LPS of H. pylori strain P466 carried the cancer-associated type-2 sialyl Lex blood-group antigen, and the LPS from strain UA915 expressed a type-1 Leb blood-group unit. These findings should aid investigations that focus on identifying and characterizing genes responsible for LPS biosynthesis in genomic strains 26695 and J99, and in understanding the role of H. pylori LPS in animal model studies. The LPSs from the H. pylori strains studied to date were grouped into specific glycotype families.

The availability of two complete Helicobacter pylori genome sequences and recent studies of its population genetics have provided a detailed picture of genetic diversity in this important human gastric pathogen. It is believed that, in addition to genetic recombination, de novo mutation could have a role in generating the high level of genetic variation in H. pylori.

Background: Helicobacter pylori infection induces autoantibodies that cross-react with human gastric mucosa from infected individuals. Candidates for the antigens responsible for molecular mimicry causing autoreactivity include the heat-shock protein HspB (Hsp60, sometimes called Hsp54) or Lewis x and Lewis y carbohydrate antigens., Objective: Our goal was to investigate the involvement of HspB (Hsp60) in autoreactivity between H. pylori and gastric biopsy tissue., Materials and Methods: Immunoelectron microscopy was used to study cross-reactivity among biopsy tissues from a patient with gastritis, gastric ulcer, and duodenal ulcer and his own serum as well as reactivity with serum raised against HspB from H. pylori and monoclonal antibodies against Lewis antigens., Results: The patient serum reacted with gastric mucosa, and the antibodies involved were predominantly IgG. Antibody raised to H. pylori HspB (Hsp60) reacted only with H. pylori cells but not with gastric mucosal tissue. In contrast, monoclonal antibodies specific for Lewis x and Lewis y antigens reacted with both H. pylori and human gastric epithelial tissue., Conclusions: Hsp60 (Hsp54) is unlikely to be involved in autoreactivity seen in individuals infected with H. pylori. In contrast, we could not rule out the role of Lewis x and Lewis y carbohydrate antigens, expressed as a component of H. pylori lipopolysaccharides, in molecular mimicry and autoantibody production.

The availability of the complete genome sequence of Helicobacter pylori 26695 has opened new avenues for research in the molecular biology of this gastric pathogen. The present review gives a general overview of H pylori obtained from the complete genome sequence and compares this with data previously obtained from cloning and functional studies of H pylori. The cagA pathogenicity island of 40 kilobases, which encodes a type IV secretion system, is discussed. The diversity of H pylori genomes is well known, yet new data indicate that some aspects of the genome, particularly outer membrane protein genes, are conserved. Genes encoding proteins involved in molecular mimicry between bacterium and gastric epithelial tissue, specifically those encoding Lewis X and Lewis Y antigens, are discussed. The large number of DNA restriction and modification genes and their role in H pylori infection are considered. Finally, gene transfer is discussed. The availability of the complete genome sequence of H pylori 26695 and the soon to be available sequence of J99 will speed up and assist in the analysis of H pylori genes and their encoded proteins. The genomes of both strains will be useful as references with which other H pylori genomes can be compared.

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

Helicobacter pylori lipopolysaccharides (LPS) express human oncofetal antigens Lewis X and Lewis Y. The synthesis of Lewis Y involves the actions of alpha (1,3) and alpha (1,2) fucosyltransferases (FucTs). Here, we report the molecular cloning and characterization of genes encoding H. pylori alpha (1,2) FucT (Hp fucT2) from various H. pylori strains. We constructed Hp fucT2 knock-out mutants and demonstrated the loss of Lewis Y production in these mutants by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The Hp fucT2 gene contains a hypermutable sequence [poly (C) and TAA repeats], which provides a possibility of frequent shifting into and out of coding frame by a polymerase slippage mechanism. Thus, the Hp fucT2 gene displays two major genotypes, consisting of either a single full-length open reading frame (ORF; as in the strain UA802) or truncated ORFs (as in the strain 26695). In vitro expression of Hp fucT2 genes demonstrated that both types of the gene have the potential to produce the full-length protein. The production of the full-length protein by the 26695 fucT2 gene could be attributed to translational-1 frameshifting, as a perfect translation frameshift cassette resembling that of the Escherichia coli dnaX gene is present. Examination of the strain UA1174 revealed that its fucT2 gene has a frameshifted ORF at the DNA level, which cannot be compensated by translation frameshifting, accounting for its Lewis Y off phenotype. In another strain, UA1218, the fucT2 gene is apparently turned off because of the loss of its promoter. Based on these data, we proposed a model for the variable expression of Lewis Y by H. pylori, in which regulation at the level of replication slippage (mutation), transcription and translation of the fucT2 gene may all be involved.

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.

About half of the world's population carries Helicobacter pylori, a gram-negative, spiral bacterium that colonizes the human stomach. The link between H. pylori and, ulceration as well as its association with the development of both gastric cancer and mucosa-associated lymphoid tissue lymphoma in humans is a serious public health concern. The publication of the genome sequences of two stains of H. pylori gives rise to direct evidence on the genetic diversity reported previously with respect to gene organization and nucleotide variability from strain to strain. The genome size of H. pylori strain 26695 is 1,6697,867 bp and is 1,643,831 bp for strain J99. Approximately 89% of the predicted open reading frames are common to both of the strains, confirming H. pylori as a single species. A region containing approximately 45% of H. pylori strain-specific open reading frames, termed the plasticity zone, is present on the chromosomes, verifying that some strain variability exists. Frequent alteration of nucleotides in the third position of the triplet codons and various copies of insertion elements on the individual chromosomes appear to contribute to distinct polymorphic fingerprints among strains analyzed by restriction fragment length polymorphisms, random amplified polymorphic DNA method, and repetitive element-polymerase chain reaction. Disordered chromosomal locations of some genes seen by pulsed-field gel electrophoresis are likely caused by rearrangement or inversion of certain segments in the genomes. Cloning and functional characterization of the genes involved in acidic survival, vacuolating toxin, cag-pathogenicity island, motility, attachment to epithelial cells, natural transformation, and the biosynthesis of lipopolysaccharides have considerably increased our understanding of the molecular genetic basis for the pathogenesis of H. pylori. The homopolymeric nucleotide tracts and dinucleotide repeats, which potentially regulate the on- and off-status of the target genes by the strand-slipped mispairing mechanism, are often found in the genes encoding the outer-membrane proteins, in enzymes for lipopolysaccharide synthesis, and within DNA modification/restriction systems. Therefore, these genes may be involved in the H. pylori-host interaction.

Background & Aims: Lewis antigens are expressed by both human gastric epithelial tissue and Helicobacter pylori. We examined Lewis antigens expressed by gastric epithelium and by H. pylori isolated from the corresponding biopsy tissue., Methods: H. pylori Lewis expression was determined by enzyme immunoassays, and immunoelectron microscopy was used to confirm the Lewis antigens on some H. pylori cells and in some biopsy specimens. Histopathology using identical monoclonal antibodies specific for Lewis A, B, X, and Y antigens was used to detect these antigens in 24 gastric biopsy specimens., Results: We identified Lewis Y in 100%, Lewis X and Lewis B in 95.8%, and Lewis A in 87.5% of biopsy specimens. In H. pylori, 87.5% expressed Lewis Y, 79.2% Lewis X, and 4.2% (one strain) Lewis B. No Lewis A was detected. Antibody specific for Lewis X labeled the bacteria and associated adhesion pedestal. The cagA gene was present in 92% of strains., Conclusions: There was no direct relationship between Lewis antigen expression by H. pylori and gastric epithelial cells in infected patients. Expression of Lewis X and Lewis Y by H. pylori suggests the possibility of their requirement for establishment and/or maintenance of infection. An immunoelectron micrograph of H. pylori interaction with the gastric epithelial adhesion pedestal suggests a tentative role for Lewis X in the adhesion process.

Clarithromycin-susceptible and clarithromycin-resistant Helicobacter pylori isolates from the same patient were investigated for the mode of development and mechanism of clarithromycin resistance. The clarithromycin-resistant strain UA1182 harbors homozygous A-to-G mutations at position 2143 in both copies of the 23S rRNA gene and has a phenotype of resistance to clarithromycin and clindamycin but no significant resistance to streptogramin B. Pulsed-field gel electrophoresis patterns of NruI- and NotI-digested genomic DNA from the Clas and Clar isolates demonstrated that they are genetically distinct, suggesting that the development of clarithromycin resistance is not from the mutation of the existing Clas strain but from a completely new strain.

Clarithromycin resistance in Helicobacter pylori is mainly due to A-to-G mutations within the peptidyltransferase region of the 23S rRNA. In the present study, cross-resistance to macrolide, lincosamide, and streptogramin B (MLS) antibiotics (MLS phenotypes) has been investigated for several clinical isolates of H. pylori. Two major types of MLS resistance were identified and correlated with specific point mutations in the 23S rRNA gene. The A2142G mutation was linked with high-level cross-resistance to all MLS antibiotics (type I), and the A2143G mutation gave rise to an intermediate level of resistance to clarithromycin and clindamycin but no resistance to streptogramin B (type II). In addition, streptogramin A and streptogramin B were demonstrated to have a synergistic effect on both MLS-sensitive and MLS-resistant H. pylori strains. To further understand the mechanism of MLS resistance in H. pylori, we performed in vitro site-directed mutagenesis (substitution of G, C, or T for A at either position 2142 or 2143 of the 23S rRNA gene). The site-directed point mutations were introduced into a clarithromycin-susceptible strain, H. pylori UA802, by natural transformation followed by characterization of their effects on MLS resistance in an isogenic background. Strains with A-to-G and A-to-C mutations at the same position within the 23S rRNA gene had similar levels of clarithromycin resistance, and this level of resistance was higher than that for strains with the A-to-T mutation. Mutations at position 2142 conferred a higher level of clarithromycin resistance than mutations at position 2143. All mutations at position 2142 conferred cross-resistance to all MLS antibiotics, which corresponds to the type I MLS phenotype, whereas mutations at position 2143 were associated with a type II MLS phenotype with no resistance to streptogramin B. To explain that A-to-G transitions were predominantly observed in clarithromycin-resistant clinical isolates, we propose a possible mechanism by which A-to-G mutations are preferentially produced in H. pylori.

Previous structural investigations performed on the lipopolysaccharides (LPSs) from the human gastric pathogen Helicobacter pylori have revealed that these cell surface glycan molecules express type 2 partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine O antigen chains (O-chains) of various lengths, which may or may not be terminated at the nonreducing end by Lewis X (Lex) and/or Ley blood group epitopes in mimicry of human cell surface glycoconjugates and glycolipids. Subsequently, serological experiments with commercially available Lewis-specific monoclonal antibodies also have recognized the presence of Lex and Ley blood group antigens in H. pylori but, in addition, have indicated the presence of type 1 chain Lea, Leb, and Led (H-type 1) blood group epitopes in some H. pylori strains. To confirm their presence, structural studies and additional serological experiments were undertaken on H. pylori strains suspected of carrying type 1 chain epitopes. These investigations revealed that the O-chain region of H. pylori strain UA948 carried both Lea (type 1) and Lex (type 2) blood group determinants. The O-chain from H. pylori UA955 LPS expressed the terminal Lewis disaccharide (type 1 chain) and Lex and Ley antigens (type 2). The O-chain of H. pylori J223 LPS carried the type 1 chain precursor Lec, the H-1 epitope (Led, type 1 chain) and an elongated nonfucosylated type 2 N-acetyllactosamine chain (i antigen). Thus, O-chains from H. pylori LPSs can also express fucosylated type 1 sequences, and the LPS from a single H. pylori strain may carry O-chains with type 1 and 2 Lewis blood groups simultaneously. That monoclonal antibodies putatively specific for the Leb determinant can detect glycan substructures (Le disaccharide, Lec, and Led) of Leb indicates their nonspecificity. The expression of both type 1 and 2 Lewis antigens by H. pylori LPSs mimics the cell surface glycomolecules present in both the gastric superficial (which expresses mainly type 1 determinants) and the superficial and glandular epithelium regions (both of which express predominantly type 2 determinants). Therefore, each H. pylori strain may have a different niche within the gastric mucosa, and each individual LPS blood group antigen may have a dissimilar role in H. pylori adaptation.

The incidence of antibiotic resistance to amoxicillin, clarithromycin, erythromycin, metronidazole and tetracycline in Helicobacter pylori strains isolated from gastric biopsy specimens obtained in Alberta was investigated. Results for all antibiotics were obtained using agar dilution, and in addition to metronidazole, the E test was used. Resistance to amoxicillin and tetracycline was not detected. Metronidazole resistance determined using agar dilution was approximately 12% (95% CI 4% to 26%) when minimal inhibitory concentrations (MICs) were at least 8 micrograms/mL, but fell to 2% (95% CI 0.1% to 13%) when MICs were set at 32 micrograms/mL or greater. The E test for metronidazole resistance (MIC 8 micrograms/mL or greater) yielded a slightly higher percentage of resistant strains compared with agar dilution tests (14%, 95% CI 5% to 29%). One of the 31 strains was resistant to clarithromycin (MIC 8 micrograms/mL) and erythromycin (MIC 16 micrograms/mL). Thus, the incidence of resistance to clarithromycin, part of the currently used triple therapy for eradication of H pylori, was 3% (95% CI 0.1% to 17%).

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to be composed of an elongated type 2 N -acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1-]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D-GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O-chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.

The genome of H. pylori is 1.68-1.73 Mb in size and contains a relatively low GC content (an average of 32.5 mol%). Physical and genetic maps of five H. pylori strains (NCTC 11637, NCTC 11638, NCTC 11639, UA 802 and UA 861) have been constructed and the complete genome sequence of strain 26695 has been determined. At least 50 genes, some of which play important roles in the physiology and pathogenicity of the bacterium, have been cloned. Marked genomic sequence variability has evolved from stain to strain demonstrated by random arrangement of 17 known genes on the chromosome and frequent mutations within individual genes. Based on such variability, sensitive and efficient molecular tying techniques such ribotyping, AR-PCR, PCR-RFLP, PCR-DNA sequencing and PFGE-RFLP have been developed and widely applied in both epidemiological and clinical studies of this pathogen. Subtypes of vacA (encoding a vacuolating cytotoxin) and the intermediate forms of a pathogenicity island (the cag region) have been identified in different H. pylori strains and these individual vacA subtypes are associated with specific clinical manifestations of H. pylori infection. Further studies on relationships between the genetic diversity and pathogenicity of H. pylori strains would lead to the development of novel and efficient therapeutic strategies for eradication of this microorganism.

In this study, we cloned and sequenced the Helicobacter pylori genes encoding fumarate reductase (FRD). H. pylori frdA, frdB and frdC specify polypeptides of 715, 245 and 254 aa, respectively. The deduced aa sequences of FrdA and FrdB are highly homologous to those of the corresponding subunits of Wolinella succinogenes FRD and also exhibit a significant sequence identity with other bacterial FRD and succinate dehydrogenase subunits A and B. However, H. pylori FrdC shares a striking degree of sequence identity only with W. succinogenes FrdC, which is a cytochrome b with two haem groups. The products encoded by H. pylori frdA, frdB and frdC were overproduced in maxicells and H. pylori FrdA was characterized using an anti-E. coli FrdA serum. H. pylori FRD activity, which was measured as fumarate-dependent benzyl viologen oxidation, is membrane-associated. Inactivation of frdA led to the loss of such activity and the mutant H. pylori cells were delayed (approx. 10-20 h behind their parent cells) in entering the mid-log phase, suggesting that FRD-driven metabolism plays an active but non-essential role for growth of H. pylori cells in vitro. H. pylori FRD contains three subunits, of which FrdA and FrdB appear to form the catalytic dimer, whereas FrdC serves as a membrane anchor.

In this study, two identical copies of a 23S-5S gene cluster, which are separately situated within the Helicobacter pylori UA802 chromosome, were cloned and sequenced. Comparison of the DNA sequence of the H. pylori 23S rRNA gene with known sequences of other bacterial 23S rRNA genes indicated that the H. pylori UA802 23S rRNA genes are closely related to those of Campylobacter spp. and therefore belong in the proposed Proteobacteria subdivision. The 5'-terminal nucleotide T or A of the 23S rRNA is close to a Pribnow box which could be a -10 region of the transcription promoter for the 23S rRNA gene, suggesting that a posttranscriptional process is likely not involved in the maturation of the H. pylori 23S rRNA. Clinical isolates of H. pylori resistant to clarithromycin were examined by using natural transformation and pulsed-field gel electrophoresis. Cross-resistance to clarithromycin and erythromycin, which was transferred by natural transformation from the Cla(r) Ery(r) donor strain H. pylori E to the Cla(s) Ery(s) recipient strain H. pylori UA802, was associated with an single A-to-G transition mutation at position 2142 of both copies of the 23S rRNA in UA802 Cla(r) Ery(r) mutants. The transformation frequency for Cla(r) and Ery(r) was found to be approximately 2 x 10(-6) transformants per viable cell, and the MICs of both clarithromycin and erythromycin for the Cla(r) Ery(r) mutants were equal to those for the donor isolate. Our results confirmed the previous findings that mutations at positions 2142 and 2143 of the H. pylori 23S rRNA gene are responsible for clarithromycin resistance and suggest that acquisition of clarithromycin resistance in H. pylori could also result from horizontal transfer.

Background: Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes. Point mutations in some genes, such as the vacA and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H. pylori strains. Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H. pylori strains., Materials and Methods: The copP gene from H. pylori UA802 was used as a model for developing a mutation screening method. Four point mutations were introduced into the copP gene by in vitro site-directed mutagenesis and were verified by DNA sequencing. The mutated copP gene replaced the wild-type locus by natural transformation and homologous recombination. The site-specific mutants were screened by polymerase chain reaction (PCR) using 3'-end mismatched primers. The origins of the PCR fragments were demonstrated by Southern hybridization with the copP-derived DNA probe., Results: Three of these four mutations were characterized by PCR with the specific primers that contained the 3'-terminal nucleotide complementary only to the mutated nucleotide on both plasmid and chromosomal DNA templates. One mutation was able to be identified with the foregoing primer containing an additional wild-type nucleotide at its 3'-end. Point mutant screening with these specific primers offers 100% sensitivity in the aforementioned conditions. To achieve optimal screening, the concentration of magnesium and the annealing temperature have to be adjusted., Conclusion: The procedure reported in this study is a simple, economical, rapid, and efficient approach in the identification of site-specific mutations on both plasmids and chromosomal DNA. Although the method was developed by using a specified H. pylori gene, it can be extended easily to other genes of interest in H. pylori or other organisms.

Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and is also associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X (Lex) and Lewis Y. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals. In this study, we report the cloning of a novel alpha1,3-fucosyltransferase gene (HpfucT) involved in the biosynthesis of Lex within H. pylori. The deduced amino acid sequence of HpfucT consists of 478 residues with the calculated molecular mass of 56,194 daltons, which is approximately 100 amino acids longer than known mammalian alpha1,3/1,4-fucosyltransferases. The approximately 52-kDa protein encoded by HpfucT was expressed in Escherichia coli CSRDE3 cells and gave rise to alpha1,3-fucosyltransferase activity but neither alpha1,4-fucosyltransferase nor alpha1,2-fucosyltransferase activity as characterized by radiochemical assays and capillary zone electrophoresis. Truncation of the C-terminal 100 amino acids of HpFuc-T abolished the enzyme activity. An approximately 72-amino acid region of HpFuc-T exhibits significant sequence identity (40-45%) with the highly conserved C-terminal catalytic domain among known mammalian and chicken alpha1,3-fucosyltransferases. These lines of evidence indicate that the HpFuc-T represents the bacterial alpha1,3-fucosyltransferase. In addition, several structural features unique to HpFuc-T, including 10 direct repeats of seven amino acids and the lack of the transmembrane segment typical for known eukaryotic alpha1,3-fucosyltransferases, were revealed. Notably, the repeat region contains a leucine zipper motif previously demonstrated to be responsible for dimerization of various basic region-leucine zipper proteins, suggesting that the HpFuc-T protein could form dimers.

The Helicobacter pylori pss gene, coding for phosphatidylserine synthase (PSS), was cloned and sequenced in this study. A polypeptide of 237 amino acids was deduced from the PSS sequence. H. pylori PSS exhibits significant amino acid sequence identity with the PSS proteins found in the archaebacterium Methanococcus jannaschii, the gram-positive bacterium Bacillus subtilis, and the yeast Saccharomyces cerevisiae but none with its Escherichia coli counterpart. Expression of the putative pss gene in maxicells gave rise to a product of approximately 26 kDa, which is in agreement with the predicted molecular mass of 26,617 Da. A manganese-dependent PSS activity was found in the membrane fractions of the E. coli cells overexpressing the H. pylori pss gene product. This result indicates that this enzyme is a membrane-bound protein, a conclusion which is supported by the fact that the PSS protein contains several local hydrophobic segments which could form transmembrane helices. The pss gene was inactivated with a chloramphenicol acetyltransferase cassette on the plasmid. However, an isogenic pss gene-disrupted mutant of H. pylori UA802 could not be obtained, suggesting that this enzyme plays an essential role in the growth of this organism.

Background: The reference strains NCTC11637 and NCTC11638 were among the very first Helicobacters ever cultured and have been distributed through national reference culture collections to researchers throughout the world. Because H. pylori is an extremely diverse species, such reference strains are invaluable as universal standards, provided that they are identified correctly., Materials and Methods: H. pylori strains (previously called "NCTC11637") from three different sources and NCTC11638 were fingerprinted by the arbitrarily primed polymerase chain reaction (PCR) (also known as random amplified polymorphic DNA, or RAPD) method and further were characterized by NotI digestion and pulsed field gel electrophoresis of total genomic DNA (NotI-PFGE) and by restriction of PCR-amplified ureCD and flaA gene segments., Results: RAPD tests of two "NCTC11637" strains from different sources (CCUG17874, UA1178) indicated that they were closely related or identical to NCTC11638. Given the diversity of H. pylori strains and the high sensitivity of the RAPD method, close matches in RAPD patterns from independent clinical isolates are not expected. In contrast, the version of "NCTC11637" from the American Type Culture Collection (ATCC43504) did not match NCTC11638 in RAPD fingerprint. Concordant results were obtained by NotI-PFGE and by restriction of PCR amplified gene segments., Conclusions: Two unrelated versions of the reference (type) H. pylori strain NCTC11637 are in general circulation and are distinguished easily by DNA fingerprinting. One matches another reference strain, NCTC11638, whereas the other is distinct from it, as expected of independent clinical isolates. Knowing which "NCTC11637" reference strain one has could be important, especially because H. pylori strains probably are diverse in phenotypic traits that are important for colonization or disease.

We previously reported that a gene hpcopA isolated from Helicobacter pylori is associated with copper transport. In the present study, the DNA upstream of the hpcopA was cloned and the nucleotide sequence analyzed. An open reading frame coding for 124 amino acids was predicted, which was connected in frame to the hpcopA. The deduced protein sequence exhibits striking homology with known copper-transporting P-type ATPases. Disruption of this ORF renders H. pylori hypersensitive to copper present in growth media, indicating that it encodes the N-terminal amino acid sequence of the hpCopA protein. Measurement of copper content in the wild-type and hpcopA-disrupted cells showed that the copper content was increased in the mutant cells, further supporting that the previous proposal that the gene hpcopA is involved in copper export. In addition, the cop operon consisting of the genes hpcopA and hpcopP was identified by primer extension and reverse transcription-polymerase chain reaction. Our results indicate that the genes for copper import and export are located in separate operons within H. pylori.

In this study, we isolated and sequenced a Helicobacter pylori gene, designated ftsH, coding for a 632-amino-acid protein which displayed striking similarity throughout its full length to FtsH proteins identified in Escherichia coli, Lactococcus lactis, and Bacillus subtilis. H. pylori FtsH also possessed approximately 200-amino-acid region containing a putative ATPase module which is conserved among members of the AAA protein family (AAA, ATPase associated with diverse cellular activities). The H. pylori ftsH product was overexpressed in E. coli and reacted immunologically with an anti-E. coli FtsH serum (T. Tomoyasu, K. Yamanaka, K. Murata, T. Suzaki, P. Bouloc, A. Kato, H. Niki, S. Hiraga, and T. Ogura, J. Bacteriol. 175:1352-1357, 1993). FtsH was also shown to be present in the membrane fraction of H. pylori, suggesting that it is membrane bound. Disruption of the ftsH gene led to the loss of viability of H. pylori, demonstrating that this gene is essential for cell growth. Overproduction of both H. pylori FtsH and E. coli FtsH together tremendously reduced the growth rate of the E. coli host cells, whereas the growth of the E. coli cells carrying the wild-type E. coli ftsH operon on the chromosome was not significantly affected by overproduction of H. pylori FtsH itself. This result suggests that the abnormal growth of cells results from interaction between H. pylori FtsH and E. coli FtsH.

Background: Helicobacter pylori is a causative pathogen of such human stomach diseases as chronic type B gastritis, ulcer, and possibly gastric carcinoma. As a cofactor in various redox enzymes and an essential trace metal required for the synthesis of metalloproteins, copper might play a role in the pathogenesis of H. pylori. A gene, copA, associated with copper transport, has been isolated from H. pylori UA802. In this study, conservation and diversity of this gene were analyzed among some Helicobacter and Campylobacter species., Materials and Methods: Twenty-one clinical isolates and strains of helicobacters and campylobacters were used in this study. Methods including polymerase chain reaction (PCR) amplification, restriction fragment-length polymorphisms (RFLPs), and hybridization were employed to carry out this work., Results: The copA gene was highly conserved in all the H. pylori isolates tested (Helicobacter nemestrinae and Helicobacter felis but not in Helicobacter mustelae and the Campylobacter species), whereas the sequence downstream of the copA appears to diverge among H. pylori isolates. In addition, two restriction patterns of the PCR-amplified copA fragments from seven H. pylori isolates and H. nemestrinae were identified, and the RFLP of H. nemestrinae was identical to that of one of the H. pylori isolate group., Conclusions: The adenosine triposphatase-derived copper-transporting mechanism is employed by various H. pylori strains, H. nemestrinae, H. felis, and perhaps by other Helicobacter species. The nucleotide mutations have risen in the copA gene. It appears that there is a genetic relatedness of the copA gene to H. pylori and H. nemestrinae.

Considerable genomic microdiversity has been reported previously among Helicobacter pylori isolates. We have constructed genome maps of four unrelated H. pylori strains (NCTC11637, NCTC11639, UA802 and UA861) using pulsed-field gel electrophoresis (PFGE) with NotI and NruI, hybridization with extracted PFGE DNA fragments and probing with 17 gene probes. These strains of H. pylori were compared with a fifth unrelated H. pylori strain NCTC11638 mapped previously. Considerable diversity in gene arrangement was evident among the five H. pylori maps, and no consistent gene clustering was found. The association of only four genes, katA (catalase gene), vacA (vacuolating cytotoxin gene), hpaA (a putative adhesin gene), and pfr (bacterial ferritin gene) were generally conserved within approximately the same 25% of the genome; however, the order of these genes also varied. Our study demonstrates that macrodiversity, i.e. variability in gene order, in addition to microdiversity, is a characteristic of the H. pylori genome.

Monoclonal antibodies (MAbs) specific for Lewis X (Lex) reacted with whole cells of Helicobacter pylori NCTC11637, UA799, UA802, UA825, UA861, UA1182, and UA1206 in immunoelectron microscopy and enzyme-linked immunosorbent assay (ELISA) experiments. These MAbs have documented specificity to Lex, whereas MAbs for Lea and Leb were negative in both immunoelectron microscopy and ELISA. H. pylori coccoid forms also reacted with the MAbs, whereas the flagellum lacking the sheath showed no reactivity. The Lex structures were associated with membrane fractions in the ELISA experiments, and silver-stained sodium dodecyl sulfate-polyacrylamide gels confirmed the presence of lipopolysaccharides which reacted with the MAbs in immunoblots. Serum from an H. pylori-infected individual contained immunoglobulins which blocked the binding of the Lex MAbs, indicating that part of the host immune response to H. pylori is to the Lex structure. The ability of this gastric pathogen to mimic an oncofetal antigen (self) could explain the down regulation of anti-H. pylori T-cell response seen in H. pylori-infected individuals.

The biosynthesis of the Lewis X determinant (Gal beta 1-4 [Fuc alpha 1-3]GlcNAc beta-) in three strains of Helicobacter pylori has been investigated. Strains UA 861, UA 802 and UA 1182 contain alpha 1,3 fucosyltransferase and beta 1,4 galactosyltransferase activities that synthesize the Lewis X structure by the transfer of monosaccharides from GDP-fucose and UDP-galactose donors, respectively. The enzyme reaction products that formed were characterized by capillary zone electrophoresis and by 1H-NMR spectroscopy. The biosynthetic pathway is therefore identical to that found in humans. In the three strains, the fucosyltransferase and galactosyltransferase activities differed in various cellular fractions. Km values for their donor and acceptor substrates also differed.

The restriction endonuclease profiles of DNAs from Helicobacter pylori strains isolated from 20 patients in two or more consecutive biopsy specimens over a period of up to 2 years were analyzed by pulsed-field gel electrophoresis with NotI and NruI. H. pylori strains possess a high degree of genomic diversity which was not observed to occur in vivo, and attempts to observe it in vitro were not successful.

A 2.7 kb fragment of Helicobacter pylori UA802 chromosomal DNA was cloned and sequenced. Three open reading frames (designated ORF1, ORF2 and ORF3, respectively) were predicted from the DNA sequence, of which ORF1 and ORF2 appeared to be located within the same operon. The deduced 611-amino-acid sequence of ORF1, a P-type ATPase (designated hpCopA), had striking homology (29-38%) with several bacterial P-type ATPase and contained the potential functional domains conserved in P-type ATPases from various sources ranging from bacterial to human. A protein of 66 amino acids (designated hpCopP) encoded by ORF2 shared extensive sequence similarity with MerP, a periplasmic mercuric ion-transporting protein, and contains the heavy metal-binding motif. Disruption of ORF1 with a chloramphenicol-resistance cassette (CAT) rendered the H. pylori mutants more susceptible to cupric ion than their parental strains, whereas there is no significant alteration of susceptibility to Ni2+, Cd2d+ and Hg2+ between the mutants and the parental strains. The results obtained indicate that ORF1 and ORF2 comprise a cation-transporting system which is associated with copper export out of the H. pylori cells.

Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragment as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site. The mass of the deduced 154-residue mature protein was 16,865 Da. Growth of E. coli cells expressing the cloned H. pylori lpp20 gene in the presence of [3H]palmitic acid resulted in radiolabelled Lpp20 while treatment of the E. coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.

Whole-cell protein extracts of Helicobacter pylori strains were evaluated by enzyme-linked immunosorbent assay to detect immunoglobulin G antibody against H. pylori in 113 patients with upper gastrointestinal complaints. These antigen preparations were of value for detecting infection by H. pylori in patients with high antibody titers (> or = 12,800), whereas for patients with lower titers, the results were inconclusive.

Most strains of Helicobacter pylori are naturally competent for uptake of chromosomal DNA. Transformation frequencies for streptomycin resistance or rifampicin resistance markers ranged from 1 x 10(-4) to 1 x 10(-3) per viable cell using a plate transformation procedure. Transformation of a metronidazole resistance marker (MtrR) was demonstrated when either a laboratory-derived mutant or a MtrR clinical isolate were used as the source of donor DNA. MtrR was transformed at a frequency of 3 x 10(-5) per viable cell. All H. pylori strains tested produce large amounts of DNAase, which may reduce DNA available for transformation. Four H. pylori plasmids were isolated. DNA fragments from H. pylori plasmids were deleted or rearranged when cloned in pUC19 and propagated in Escherichia coli DH5 alpha. An H. pylori plasmid, pUOA26 which contained a chloramphenicol resistance determinant from Campylobacter coli, was constructed in H. pylori. This plasmid could be successfully introduced by natural transformation only into H. pylori recipients which contained a homologous resident plasmid. Transformation of pUOA26 into plasmid-free cells of H. pylori was achieved by electroporation. Transformation frequencies were 1 x 10(-4) transformants per viable cell when plasmid DNA was isolated from the same strain; however, introduction of pUOA26 DNA derived from H. pylori 8091 into a different H. pylori strain, NCTC 11639, resulted in transformation at much lower frequencies (< or = 1 x 10(-7) per viable cell).(ABSTRACT TRUNCATED AT 250 WORDS)

Genomic DNA from 30 strains of Helicobacter pylori was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with NotI and NruI. The genome sizes of the strains ranged from 1.6 to 1.73 Mb, with an average size of 1.67 Mb. By using NotI and NruI, a circular map of H. pylori UA802 (1.7 Mb) which contained three copies of 16S and 23S rRNA genes was constructed. An unusual feature of the H. pylori genome was the separate location of at least two copies of 16S and 23S rRNA genes. Almost all strains had different PFGE patterns after NotI and NruI digestion, suggesting that the H. pylori genome possesses a considerable degree of genetic variability. However, three strains from different sites (the fundus, antrum, and body of the stomach) within the same patient gave identical PFGE patterns. The genomic pattern of individual isolates remained constant during multiple subcultures in vitro. The reason for the genetic diversity observed among H. pylori strains remains to be explained.