Ecological succession in the vaginal microbiota during pregnancy and birth.

The mother's vaginal microbiota represents the first microbes to which a child is exposed when delivered vaginally. However, little is known about the composition and development of the vaginal microbiota during pregnancy and birth. Here, we analyzed the vaginal microbiota of 57 women in pregnancy week 24, 36 and at birth after rupture of membranes but before delivery, and further compared the composition with that of the gut and airways of the 1-week-old child. The vaginal community structure had dramatic changes in bacterial diversity and taxonomic distribution, yet carried an individual-specific signature. The relative abundance of most bacterial taxa increased stepwise from week 24 of pregnancy until birth, with a gradual decline of Lactobacillus. Mother-to-child vertical transfer, as suggested by sharing, was modest, with the strongest transfer being for Clostridiales followed by Lactobacillales and Enterobacteriales. In conclusion, late gestation is associated with an increase in maternal vaginal microbiota diversity, and vaginal bacteria at birth only modestly predict the composition of the neonatal microbiota.

Salivary dysbiosis and the clinical spectrum in anti-Ro positive mothers of children with neonatal lupus.

Mothers giving birth to children with manifestations of neonatal lupus (NL) represent a unique population at risk for the development of clinically evident pathologic autoimmunity since many are asymptomatic and only become aware of anti-SSA/Ro positivity (anti-Ro+) based on heart block in their fetus. Accordingly, we hypothesized that the microbiome in saliva is associated with the development of autoreactivity and in some cases the progression in health status from benign to overt clinical disease including Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The study comprised a clinical spectrum of anti-Ro+ mothers, all of whom gave birth to a child with NL: 9 were asymptomatic or had an undifferentiated autoimmune disease (Asym/UAS) and 16 fulfilled criteria for SS and/or SLE. Microbial diversity was reduced across all levels from kingdom to species for the anti-Ro+ mothers vs healthy controls; however, there were no significant differences between Asym/UAS and SS/SLE mothers. Relative abundance of Proteobacteria and more specifically class Betaproteobacteria decreased with clinical severity (healthy controls < Asym/UAS < SS/SLE). These ordered differences were maintained through the taxonomic hierarchy to three genera (Lautropia, Comamonas, and Neisseria) and species within these genera (L. mirabilis, N. flavescens and N. oralis). Biometric analysis comparing von Willebrand Factor domains present in human Ro60 with L. mirabilis proteins support the hypothesis of molecular mimicry. These data position the microbiome in the development of anti-Ro reactivity and subsequent clinical spectrum of disease.

Gut microbiome of treatment-naïve MS patients of different ethnicities early in disease course.

Although the intestinal microbiome has been increasingly implicated in autoimmune diseases, much is unknown about its roles in Multiple Sclerosis (MS). Our aim was to compare the microbiome between treatment-naïve MS subjects early in their disease course and controls, and between Caucasian (CA), Hispanic (HA), and African American (AA) MS subjects. From fecal samples, we performed 16S rRNA V4 sequencing and analysis from 45 MS subjects (15 CA, 16 HA, 14 AA) and 44 matched healthy controls, and whole metagenomic shotgun sequencing from 24 MS subjects (all newly diagnosed, treatment-naïve, and steroid-free) and 24 controls. In all three ethnic groups, there was an increased relative abundance of the same single genus, Clostridium, compared to ethnicity-matched controls. Analysis of microbiota networks showed significant changes in the network characteristics between combined MS cohorts and controls, suggesting global differences not restricted to individual taxa. Metagenomic analysis revealed significant enrichment of individual species within Clostridia as well as particular functional pathways in the MS subjects. The increased relative abundance of Clostridia in all three early MS cohorts compared to controls provides candidate taxa for further study as biomarkers or as etiologic agents in MS.

Association of prenatal antibiotics with foetal size and cord blood leptin and adiponectin.

BACKGROUND: Early postnatal antibiotic use has been shown to promote excess weight gain, but it is unclear whether intrauterine exposure to antibiotics is associated with foetal growth and adiposity. The objective of this study was to examine associations of antibiotic prescription in each trimester of pregnancy with foetal size and adipokine levels at birth. METHODS: In 2128 pregnant women from the pre-birth Project Viva cohort, from electronic medical records, we estimated antibiotic prescribing by timing during pregnancy. Outcomes were sex-specific birth weight-for-gestational-age z-score (BW/GA-z) and levels of umbilical cord leptin and adiponectin. We used linear regression models adjusted for maternal age, pre-pregnancy body mass index, parity, race/ethnicity, education, smoking during pregnancy, household income and child sex and additionally adjusted cord blood leptin and adiponectin models for gestation length. RESULTS: Of the 2128 women in our sample, 643 (30.2%) were prescribed with oral antibiotics during pregnancy. Mean (standard deviation) BW/GA-z was 0.17 (0.97), cord blood leptin was 9.0 ng mL-1 (6.6) and cord blood adiponectin was 28.8 ng mL-1 (6.8). Overall, antibiotic prescription in pregnancy was associated with lower BW/GA-z [multivariable adjusted ß -0.11; 95% confidence interval {CI} -0.20, -0.01]. In trimester-specific analyses, only second trimester antibiotic prescription was associated with lower BW/GA-z (ß -0.23; 95% CI -0.37, -0.08). Overall, antibiotic prescription in pregnancy was not associated with cord blood leptin or adiponectin levels. However, in trimester-specific analyses, third trimester antibiotic prescription was associated with higher cord blood leptin (ß 2.28 ng mL-1 ; 95% CI 0.38, 4.17). CONCLUSIONS: Antibiotics in mid-pregnancy were associated with lower birth weight for gestational age, whereas third trimester antibiotics were associated with higher cord blood leptin.

Helicobacter pylori in children with asthmatic conditions at school age, and their mothers.

BACKGROUND: Helicobacter pylori prevalence in Western countries has been declining simultaneously with increases in childhood asthma and allergic diseases; prior studies have linked these phenomena. AIMS: To examine the association between H. pylori colonisation in children and risk of asthma and related conditions at school age. We secondly examined additional effects of maternal H. pylori status by pairing with children's status. METHODS: This study was embedded in a multi-ethnic population-based cohort in Rotterdam, The Netherlands. We measured anti-H. pylori and anti-CagA antibodies in serum of children obtained at age 6 years, and of their mothers obtained during midpregnancy. Asthma or related conditions were reported for children at age 6 years. We used multivariate logistic regression analyses among 3797 subjects. RESULTS: In children, the H. pylori positivity rate was 8.7%, and 29.2% of these were CagA-positive. A child's colonisation with a CagA-negative-H. pylori strain was associated with an increased risk of asthma (Odds ratio 2.11; 95% CI 1.23-3.60), but this differed for European (3.64; 1.97-6.73) and non-European (0.52; 0.14-1.89) children. When taking into account maternal H. pylori status, only H. pylori-positive children with an H. pylori-negative mother had increased risk of asthma (2.42; 1.11-5.27), accounting for 3.4% of the asthma risk. CONCLUSIONS: Colonisation of a European child with a CagA-negative-H. pylori strain at age 6 was associated with an increased prevalence of asthma, but there was no association for non-European children. The underlying mechanisms for the observed risk differences require further research.

Association of caesarean delivery with child adiposity from age 6 weeks to 15 years.

OBJECTIVES: o assess associations of caesarean section with body mass from birth through adolescence. DESIGN: ongitudinal birth cohort study, following subjects up to 15 years of age. SETTING AND PARTICIPANTS: Children born in 1991-1992 in Avon, UK who participated in the Avon Longitudinal Study of Parents and Children (ALSPAC) (n=10 219). PRIMARY OUTCOME: standardized measures of body mass (weight-for length z-scores at 6 weeks, 10 and 20 months; and body mass index (BMI) z-scores at 38 months, 7, 9, 11 and 15 years). Secondary outcome: categorical overweight or obese (BMI: 85th percentile) for age and gender, at 38 months, 7, 9, 11 and 15 years. RESULTS: Of the 10 219 children, 926 (9.06%) were delivered by caesarean section. Those born by caesarean had lower-birth weights than those born vaginally (-46.1 g, 95% confidence interval(CI): 14.6-77.6 g; P=0.004). In mixed multivariable models adjusting for birth weight, gender, parental body mass, family sociodemographics, gestational factors and infant feeding patterns, caesarean delivery was consistently associated with increased adiposity, starting at 6 weeks (+0.11 s.d. units, 95% CI: 0.03-0.18; P=0.005), through age 15 (BMI z-score increment+0.10 s.d. units, 95% CI: 0.001-0.198; P=0.042). By age 11 caesarean-delivered children had 1.83 times the odds of overweight or obesity (95% CI: 1.24-2.70; P=0.002). When the sample was stratified by maternal pre-pregnancy weight, the association among children born of overweight/obese mothers was strong and long-lasting. In contrast, evidence of an association among children born of normal-weight mothers was weak. CONCLUSION: Cesarean delivery is associated with increased body mass in childhood and adolescence. Research is needed to further characterize the association in children of normal weight women. Additional work is also needed to understand the mechanism underlying the association, which may involve relatively enduring changes in the intestinal microbiome.

Infant antibiotic exposures and early-life body mass.

OBJECTIVES: To examine the associations of antibiotic exposures during the first 2 years of life and the development of body mass over the first 7 years of life. DESIGN: Longitudinal birth cohort study. SUBJECTS: A total of 11 532 children born at 2500 g in the Avon Longitudinal Study of Parents and Children (ALSPAC), a population-based study of children born in Avon, UK in 1991-1992. MEASUREMENTS: Exposures to antibiotics during three different early-life time windows (<6 months, 6-14 months, 15-23 months), and indices of body mass at five time points (6 weeks, 10 months, 20 months, 38 months and 7 years). RESULTS: Antibiotic exposure during the earliest time window (<6 months) was consistently associated with increased body mass (+0.105 and +0.083 s.d. unit, increase in weight-for-length Z-scores at 10 and 20 months, P<0.001 and P=0.001, respectively; body mass index (BMI) Z-score at 38 months +0.067 s.d. units, P=0.009; overweight OR 1.22 at 38 months, P=0.029) in multivariable, mixed-effect models controlling for known social and behavioral obesity risk factors. Exposure from 6 to 14 months showed no association with body mass, while exposure from 15 to 23 months was significantly associated with increased BMI Z-score at 7 years (+0.049 s.d. units, P=0.050). Exposures to non-antibiotic medications were not associated with body mass. CONCLUSIONS: Exposure to antibiotics during the first 6 months of life is associated with consistent increases in body mass from 10 to 38 months. Exposures later in infancy (6-14 months, 15-23 months) are not consistently associated with increased body mass. Although effects of early exposures are modest at the individual level, they could have substantial consequences for population health. Given the prevalence of antibiotic exposures in infants, and in light of the growing concerns about childhood obesity, further studies are needed to isolate effects and define life-course implications for body mass and cardiovascular risks.

Host-interactive genes in Amerindian Helicobacter pylori diverge from their Old World homologs and mediate inflammatory responses.

Helicobacter pylori is the dominant member of the gastric microbiota and has been associated with an increased risk of gastric cancer and peptic ulcers in adults. H. pylori populations have migrated and diverged with human populations, and health effects vary. Here, we describe the whole genome of the cag-positive strain V225d, cultured from a Venezuelan Piaroa Amerindian subject. To gain insight into the evolution and host adaptation of this bacterium, we undertook comparative H. pylori genomic analyses. A robust multiprotein phylogenetic tree reflects the major human migration out of Africa, across Europe, through Asia, and into the New World, placing Amerindian H. pylori as a particularly close sister group to East Asian H. pylori. In contrast, phylogenetic analysis of the host-interactive genes vacA and cagA shows substantial divergence of Amerindian from Old World forms and indicates new genotypes (e.g., VacA m3) involving these loci. Despite deletions in CagA EPIYA and CRPIA domains, V225d stimulates interleukin-8 secretion and the hummingbird phenotype in AGS cells. However, following a 33-week passage in the mouse stomach, these phenotypes were lost in isolate V225-RE, which had a 15-kb deletion in the cag pathogenicity island that truncated CagA and eliminated some of the type IV secretion system genes. Thus, the unusual V225d cag architecture was fully functional via conserved elements, but the natural deletion of 13 cag pathogenicity island genes and the truncation of CagA impaired the ability to induce inflammation.

Helicobacter pylori Genome Plasticity.

Helicobacter pylori, a Gram-negative pathogen associated with ulcers, chronic gastritis, and gastric cancers, has been a resident of the human stomach since early human history [1]. This association has only recently begun to erode with the advent of antibiotics and modern lifestyles, but even today H. pylori colonizes approximately half the world's population. To have remained a successful colonizer of humans during thousands of years of association, populations of H. pylori must have been able to survive and adapt to countless evolutionary challenges within and between hosts. As a species, H. pylori possesses one of the most fluid genomes within the prokaryotic kingdom [2], a characteristic that has likely aided its continued success. H. pylori exhibits exceptionally high rates of DNA point mutations, intragenomic recombination (facilitated by repetitive elements common in H. pylori genomes), and intergenomic recombination (mediated by natural transformation), all of which contribute to the high genomic variability between isolates. Previous reviews have focused on these processes as agents of evolutionary change within H. pylori [2-8]. The mechanisms of both mutation and natural transformation, and the evolutionary processes that retain genetic variation generated by these mechanisms, dictate the extent to which each contributes to genomic diversity in the context of different bacterial population structures [9-13]. Unlike well-studied evolutionary systems, such as Salmonella and Escherichia coli, H. pylori is notable in its lack of an environmental reservoir outside of human and other primate stomachs, suggesting that between-host survival is a relatively weak determinant of selection pressures [14, 15]. Given that H. pylori exist largely as distinct host-associated populations, it is possible to begin to model the evolutionary mechanisms that affect the long-term persistence of this species. In this chapter, we consider how the attributes of H. pylori's natural history as a long-term resident of the human stomach and the specific mechanisms of mutation and genetic exchange in this organism have shaped the H. pylori genome. We begin with a survey of genome plasticity in H. pylori. We then discuss mechanisms of mutation and natural transformation in H. pylori and examine experimental evidence for the generation of genomic changes within populations. Finally, we consider how different models of H. pylori population structure affect the relative contributions of mutation and recombination to the evolutionary success of this organism. By bridging evolutionary studies with investigations of pathogenesis from a molecular perspective, we hope to shed new light on how H. pylori has and continues to evolve with its human hosts.

The association of gastric leptin with oesophageal inflammation and metaplasia.

BACKGROUND: Gastro-oesophageal reflux disease complications may reflect imbalances between protective and injurious factors. Through its effects on cell growth, leptin may influence oesophageal mucosal homeostasis. AIMS: To determine whether leptin receptors are present in the oesophagus, and whether serum or gastric leptin levels are associated with oesophageal inflammation and metaplasia. METHODS: From patients referred for upper endoscopy, biopsies were obtained from the stomach and distal oesophagus, and serum samples were collected. Patients were classified as having normal, inflamed or Barrett's oesophagus. Quantitative immunohistochemistry was performed on representative sections, and leptin levels in plasma and gastric biopsy samples were determined by specific immunoassay. RESULTS: Of 269 individuals enrolled, 105 were Helicobacter pylori-negative. Of the 88 patients with complete oesophageal biopsies, 44 were normal, 24 were inflamed and 20 were Barrett's oesophagus. Receptors for leptin were highly expressed on oesophageal epithelial cells, with similar density and staining pattern in all three conditions, and plasma and antral leptin levels did not differ significantly. Patients with Barrett's had significantly (p = 0.01) higher fundic leptin levels (median 202 (interquartile range 123-333) pg/mg) compared with normal (126 (78-221) pg/mg) or inflamed (114 (76-195) pg/mg) oesophagus. In multivariate analysis, for every twofold increase in fundic leptin, the odds of having Barrett's was 3.4 times (95% CI 1.5 to 7.6) higher compared with having a normal oesophagus. CONCLUSIONS: Leptin receptor expression on oesophageal epithelial cells provides a pathway for leptin-mediated signal transduction. Variation in gastric leptin production could contribute to differential oesophageal healing and metaplasia progression.

Helicobacter pylori and oesophageal and gastric cancers in a prospective study in China.

In a cohort of 29,584 residents of Linxian, China, followed from 1985 to 2001, we conducted a case-cohort study of the magnitude of the association of Helicobacter pylori seropositivity with cancer risk in a random sample of 300 oesophageal squamous cell carcinomas, 600 gastric cardia adenocarcinomas, all 363 diagnosed gastric non-cardia adenocarcinomas, and a random sample of the entire cohort (N=1050). Baseline serum was evaluated for IgG antibodies to whole-cell and CagA H. pylori antigens by enzyme-linked immunosorbent assay. Risks of both gastric cardia and non-cardia cancers were increased in individuals exposed to H. pylori (Hazard ratios (HRs) and 95% confidence intervals=1.64; 1.26-2.14, and 1.60; 1.15-2.21, respectively), whereas risk of oesophageal squamous cell cancer was not affected (1.17; 0.88-1.57). For both cardia and non-cardia cancers, HRs were higher in younger individuals. With longer time between serum collection to cancer diagnosis, associations became stronger for cardia cancers but weaker for non-cardia cancers. CagA positivity did not modify these associations. The associations between H. pylori exposure and gastric cardia and non-cardia adenocarcinoma development were equally strong, in contrast to Western countries, perhaps due to the absence of Barrett's oesophagus and oesophageal adenocarcinomas in Linxian, making all cardia tumours of gastric origin, rather than a mixture of gastric and oesophageal malignancies.

High frequency of gastric colonization with multiple Helicobacter pylori strains in Venezuelan subjects.

Multiple Helicobacter pylori strains may colonize an individual host. Using enzyme-linked immunosorbent assay and line probe assay (LiPA) techniques, we analyzed the prevalence of mixed H. pylori colonization in 127 subjects from Venezuela, a country of high H. pylori prevalence, from three regions representing different population groups: the Andes (Merida), where Caucasian mestizos predominate, a major city near the coast (Caracas), where Amerindian-Caucasian-African mestizos predominate, and an Amazonian community (Puerto Ayacucho), where Amerindians predominate and mestizos reflect Amerindian and Caucasian ancestry. Among 121 H. pylori-positive persons, the prevalence of cagA-positive strains varied from 50% (Merida) to 86% (Puerto Ayacucho) by LiPA. Rates of mixed colonization also varied, as assessed by LiPA of the vacA s (mean, 49%) and m (mean, 26%) regions. In total, 55% of the individuals had genotypic evidence of mixed colonization. vacA s1c, a marker of Amerindian (East Asian) origin, was present in all three populations, especially from Puerto Ayacucho (86%). These results demonstrate the high prevalence of mixed colonization and indicate that the H. pylori East Asian vacA genotype has survived in all three populations tested.

Evolutionary history of hrgA, which replaces the restriction gene hpyIIIR in the hpyIII locus of Helicobacter pylori.

A recently identified Helicobacter pylori gene, hrgA, was previously reported to be present in 70 (33%) of 208 strains examined (T. Ando, T. M. Wassenaar, R. M. Peek, R. A. Aras, A. I. Tschumi, L.-J. Van Doorn, K. Kusugami, and M. J. Blaser, Cancer Res. 62:2385-2389, 2002). Sequence analysis of nine such strains indicated that in each strain hrgA replaced hpyIIIR, which encodes a restriction endonuclease and which, together with the gene for its cognate methyltransferase, constitutes the hpyIII locus. As a consequence of either the hrgA insertion or independent mutations, hpyIIIM function was lost in 11 (5%) of the 208 strains examined, rendering chromosomal DNA sensitive to MboI digestion. The evolutionary history of the locus containing either hpyIII or hrgA was reconstructed. By homologous recombination involving flanking sequences, hrgA and hpyIIIR can replace one another in the hpyIII locus, and there is simultaneous replacement of several flanking genes. These findings, combined with the hpyIM/iceA2 locus discovered previously, suggest that the two most strongly conserved methylase genes of H. pylori, hpyIIIM and hpyIM, are both preceded by alternative genes that compete for presence at their loci.

Role of S-layer protein antigenic diversity in the immune responses of sheep experimentally challenged with Campylobacter fetus subsp. fetus.

Surface layer proteins (SLPs) are essential for induction of abortion by Campylobacter fetus subsp. fetus in experimentally challenged ewes. These proteins are encoded by multiple sap genes and vary in size and antigenicity. The role of SLP antigenic variation during experimental ovine infection was investigated. Following subcutaneous challenge, the SLPs were highly antigenic, and antibodies were detected in serum, milk, bile, and urine. Fecal anti-SLP antibodies were detected only in animals challenged orally. Ewes challenged with wild-type strain 23D with variable SLPs developed detectable circulating anti-SLP immunoglobulin G (IgG) antibodies by 2 weeks postchallenge. In contrast, ewes challenged with mutants of 23D that had fixed expression of a single SLP developed antibodies within 1 week postchallenge, suggesting that antigenic variation in SLPs may delay the host antibody response. Although not statistically significant, the data from challenge experiments in which vaccinated ewes were used suggested that SLP-expressing vaccines could protect animals from abortion and that this effect was independent of the SLP expressed, indicating involvement of conserved epitopes in the SLP. The conserved 184-amino-acid N-terminal region of the SLP, identified from previously published sequences, was epitope mapped with rabbit anti-SLP antisera by using overlapping synthetic 20-mer peptides. Two putative epitopes were identified at amino acids 81 to 110 and 141 to 160. Amino acids 81 to 100 also bound serum IgG antibodies from experimentally challenged sheep. Conserved antigenic regions of the SLP that induce protective immune responses may enable development of synthetic vaccine candidates for C. fetus subsp. fetus-associated ovine abortion.

Evidence that cagA(+) Helicobacter pylori strains are disappearing more rapidly than cagA(-) strains.

BACKGROUND AND AIMS: The prevalence of Helicobacter pylori colonisation in populations in developed country has been declining, as shown by community based serological surveys of adults in Vammala, Finland in 1973 and 1994. In this study, we determined whether the proportion of subjects colonised by cagA(+) or cagA(-) H pylori strains has changed as the overall prevalence of H pylori(+) has declined. METHODS: We examined 911 sera from Vammala's study for antibodies to the CagA antigen of H pylori using a truncated CagA protein as the antigen in an ELISA and we examined the trend in acquisition and carriage of cagA(+) strains. RESULTS: As expected, the prevalence of carriage of both cagA(+) and cagA(-) strains fell between 1973 and 1994 (p<0.001). However, the prevalence of cagA(+) strains among those <45 years declined (34% to 8%) significantly (p<0.001) more than for cagA(-) strains (12% to 6%). Of 221 subjects with paired serum samples, 12 (5.4%) changed H pylori status; the estimated seroconversion and reversion rates were 0.4% and 0.13% per year, respectively. Except for the few individuals who changed serostatus, absolute antibody levels to H pylori antigens, including CagA, changed little over the 21 year period. CONCLUSIONS: The decline in CagA seroprevalence predominantly reflects declining acquisition of cag(+) strains in younger subjects. In addition, these data confirm that H pylori acquisition chiefly occurs during childhood but continues to occur during adulthood, albeit at low rates, in developed countries.

Dynamics of bacterial phenotype selection in a colonized host.

The population dynamics of Helicobacter pylori during colonization in an infected animal host provide a quantifiable experimental model of in vivo microbial phenotype evolution. Phenotype variability in H. pylori populations can be typed as polymorphic expression of Lewis antigens on their cell surfaces. The high mutational frequency of H. pylori for Lewis expression provides substrate for differential selection by the host. Experimental challenge and successful colonization of mice and gerbils allows tracking of H. pylori phenotype variability from the initial inoculation to the ultimate establishment of a quasispecies. Colonization data provide a quantitative experimental model of phenotype evolution in a relatively large population (>10(4) individuals) over a relatively long evolutionary time scale (>10(3) generations). A mathematical model is developed to interpret the data in terms of the dynamic processes occurring during colonization. The mathematical model distinguishes the roles of selection and mutation; quantifies the effects of initial phenotype diversity, mutational frequency, and selective advantage; and applies generally to phenotype evolution in biological populations.

Polymorphisms of Helicobacter pylori HP0638 reflect geographic origin and correlate with cagA status.

Since the associations between Helicobacter pylori genotype and disease differ in Asia and the West, we investigated the correlation between HP0638, encoding an outer membrane protein, and potential markers of virulence (cagA, vacA, and iceA). For 109 strains from nine countries, the status of cagA, vacA, and iceA was determined by PCR and/or a line probe assay. We also studied 18 strains from 8 patients (parents and 6 daughters) from a Dutch family and paired strains collected on average 8 years apart from 11 patients. When the HP0638 signal sequences were amplified by PCR and DNA sequence determinations were performed, 89 (96%) of 93 cagA-positive strains had HP0638 in frame, versus none (0%) of 16 cagA-negative strains (P < 0.001). Among strains in which HP0638 was in frame, a six-CT dinucleotide repeat pattern was dominant in Western countries (23 of 33 strains [70%]), while a pattern of three CT repeats with another CT after four T's (3 + 1-CT-repeat pattern) was dominant in East Asia (31 of 46 strains [67%]); however, specific CT repeat patterns did not correlate with clinical outcome. HP0638 phylogenetic trees also showed geographic characters. The HP0638 frame status and CT dinucleotide repeat patterns were identical for 9 of 11 pairs of strains obtained on average 8 years apart from individuals and the 15 strains obtained from the mother and all six daughters. Thus, HP0638 frame status and cagA status are strongly correlated. The CT dinucleotide repeat pattern in the putative HP0638 signal sequence has geographic characters and appears stable in particular patients and families over a period of years. Analysis of HP0638 CT polymorphisms may serve as a new typing system to discriminate H. pylori isolates for epidemiological purposes.