Brief Article Open Access
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Feb 28, 2013; 19(8): 1283-1291
Published online Feb 28, 2013. doi: 10.3748/wjg.v19.i8.1283
Ethnicity association of Helicobacter pylori virulence genotype and metronidazole susceptibility
Hanafiah Alfizah, Department of Medical Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, 56000 Cheras, Kuala Lumpur, Malaysia
Awang Hamat Rukman, Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Ahmad Norazah, Bacteriology Unit, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia
Razlan Hamizah, Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, 56000 Cheras, Kuala Lumpur, Malaysia
Mohamed Ramelah, Centre for Innovative Collaboration, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia
Author contributions: All authors contributed equally in this study.
Supported by Grant from the Ministry of Science, Technology and Innovation, Malaysia, No. 06-02-0055-PR0073/05
Correspondence to: Hanafiah Alfizah, PhD, Department of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Malaysia. alfizah@ppukm.ukm.edu.my
Telephone: +60-3-91455442 Fax: +60-3-91456671
Received: October 17, 2012
Revised: December 29, 2012
Accepted: January 11, 2013
Published online: February 28, 2013

Abstract

AIM: To characterise the cag pathogenicity island in Helicobacter pylori (H. pylori) isolates by analysing the strains’vacA alleles and metronidazole susceptibilities in light of patient ethnicity and clinical outcome.

METHODS: Ninety-five H. pylori clinical isolates obtained from patients with dyspepsia living in Malaysia were analysed in this study. Six genes in the cagPAI region (cagE, cagM, cagT, cag13, cag10 and cag67) and vacA alleles of the H. pylori isolates were identified by polymerase chain reaction. The isolates’ metronidazole susceptibility was also determined using the E-test method, and the resistant gene was characterised by sequencing.

RESULTS: More than 90% of the tested isolates had at least one gene in the cagPAI region, and cag67 was predominantly detected in the strains isolated from the Chinese patients, compared with the Malay and Indian patients (P < 0.0001). The majority of the isolates (88%) exhibited partial deletion (rearrangement) in the cagPAI region, with nineteen different patterns observed. Strains with intact or deleted cagPAI regions were detected in 3.2% and 8.4% of isolates, respectively. The prevalence of vacA s1m1 was significantly higher in the Malay and Indian isolates, whereas the isolates from the Chinese patients were predominantly genotyped as vacA s1m2 (P = 0.018). Additionally, the isolates from the Chinese patients were more sensitive to metronidazole than the isolates from the Malay and Indian patients (P = 0.047). Although we attempted to relate the cagPAI genotypes, vacA alleles and metronidazole susceptibilities to disease outcome, no association was observed. The vacA alleles were distributed evenly among the strains with intact, partially deleted or deleted cagPAI regions. Interestingly, the strains exhibiting an intact cagPAI region were sensitive to metronidazole, whereas the strains with a deleted cagPAI were more resistant.

CONCLUSION: Successful colonisation by different H. pylori genotypes is dependent on the host’s genetic makeup and may play an important role in the clinical outcome.

Key Words: Helicobacter pylori, cag pathogenicity island, vacA alleles, Metronidazole susceptibility



INTRODUCTION

Helicobacter pylori (H. pylori) demonstrates great genetic diversity, as evidenced by the apparently unlimited number of unique strains differing in genome size, gene order, gene content and allelic profile[1]. The high levels of genetic diversity in clinical isolates and the presence of certain virulence genotypes may confer a selective advantage to certain strains in different hosts. Therefore, the virulence genotype of H. pylori is a potentially useful predictor of the clinical outcome of gastric mucosal colonisation.

H. pylori possess a number of virulence determinants that modulate the organism’s interaction with a host[2,3]. These virulence factors include the secreted, vacuolating cytotoxin VacA and the gene products of the pathogenicity island (cagPAI). The presence of these genetic loci correlates with the more severe H. pylori-associated pathologies, peptic ulcers and gastric cancer. Screening for the cagPAI genes is frequently performed due to the genes’ involvement in virulence characteristics. An infection with cagPAI-positive H. pylori strains significantly increases the risk of developing severe gastric mucosal inflammation, duodenal ulceration and gastric cancer[4-6]. It has been observed that only one-half to two-thirds of isolates from Western patients express the cagPAI region, whereas almost all of the East Asian strains express this region. As there is limited information on the structure of the cagPAI in Malaysian patients, the role of cagPAI rearrangement in H. pylori isolates from various ethnic populations within this country should be investigated.

The vacA gene is another H. pylori virulence factor that exhibits great polymorphism and diverse allelic combination. The variable structure, resulting in different allelic arrangements in the gene, is related to differences in cytotoxin production and to the distinct clinical outcomes of H. pylori infection[7-9]. For example, a recent study performed at our institute has demonstrated a correlation between the presence of genetic polymorphisms in the vacA gene and the severity of gastritis[10].

H. pylori infection can be effectively cured by antibiotics. More specifically, metronidazole is commonly used in conjunction with either amoxicillin or clarithromycin and a proton-pump inhibitor to eradicate H. pylori infection[11,12]. Metronidazole is also used in the treatment of other diseases, such as gynaecological infections, and has contributed to the increased antibiotic resistance of H. pylori[13]. In Malaysia, the prevalence of metronidazole-resistant strains is 37%, which may be one of the factors contributing to the failed eradication of H. pylori infection in the country[14].

The population of Malaysia consists of three major ethnic groups (Malay, Chinese and Indian) that are historically associated with differences in H. pylori infection[15-17]. H. pylori-associated gastroduodenal diseases typically do not reflect the prevalence of H. pylori infection[15]. Furthermore, geographical and population differences in H. pylori virulence factors and disease severity underscore the importance of investigating the relationship between the genotypes of the causative strains and the clinical outcome. Therefore, to further understand the dynamics of the genetic traits of the H. pylori cagPAI regions, vacA alleles and metronidazole susceptibilities in Malaysia’s multiethnic population, we investigated the possible association between these H. pylori virulence factors and infection in patients of different ethnicities and clinical outcomes.

MATERIALS AND METHODS
Patients

Patients with dyspepsia who had undergone an upper gastrointestinal scope (OGDS) at Universiti Kebangsaan Malaysia Medical Centre (Kuala Lumpur, Malaysia) between May 2004 and December 2007 were recruited. Any patients who had received antibiotics 4 wk prior to OGDS were excluded. Written informed consent was obtained from each patient, and the protocol was approved by the Research Ethics Committee (protocol number FF-075-2003) of the University.

H. pylori culture

Biopsies were subcultured for H. pylori on Columbia agar base (Oxoid, Basingstoke, England) containing Dent’s supplement (Oxoid, Basingstoke, England) and 7% defibrinated sheep blood. The plates were incubated at 37  °C for 5 d under microaerophilic conditions.

DNA extraction

H. pylori genomic DNA was prepared using a High Pure polymerase chain reaction (PCR) Template Preparation Kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions.

Detection of metronidazole susceptibility and resistance genotype

Metronidazole susceptibility was determined using the E-test method (AB Biodisk, Solna, Sweden). First, an H. pylori culture suspension with a McFarland turbidity of 3.0 was used to inoculate Columbia agar supplemented with 10% defibrinated sheep blood. The plates were then incubated at 35 ± 2  °C for 3-5 d under microaerophilic conditions. The isolates were classified as resistant to metronidazole when the MIC value was > 8 μg/mL[18]. The rdxA genes were amplified by PCR using specific primers, as previously described[19], and then subjected to sequencing.

Detection of cagPAI genes

Two loci were selected in the cagI region (cagE and cagM), and four loci were chosen from the cagII region (cagT, cag13, cag10 and cag67). Additionally, the insertion sequence IS605 was selected. The presence of these selected genes was determined by PCR using specific primers, as previously described[20]. PCR was conducted in 25 μL volumes containing 1 × PCR buffer, 0.2 mmol/L dNTP mix, 10 pmol of each primer, 1 U Taq polymerase (BioTherm, GeneCraft, Germany) and 1 μL of the DNA sample. The PCR amplification conditions consisted of 35 cycles at 94 °C for 1 min, 50-55 °C for 30 s, 72 °C for 1 min, and one cycle for the final extension at 72 °C for 10 min. The amplified product was electrophoresed on a 1% agarose gel.

Detection of vacA alleles

The vacA genotyping was performed by multiplex PCR using specific primers, as previously described[10,21]. PCR was conducted in 25-μL volumes containing 1 × PCR buffer, 0.2 mmol/L dNTP mix, 6 pmol of each primer, 0.75 U Taq polymerase (BioTherm, GeneCraft, Germany) and 1 μL of the DNA sample. The PCR amplification conditions consisted of an initial denaturation step at 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 30 s, primer annealing at 55 °C for 30 s, extension at 72 °C for 30 s, and one cycle for the final extension at 72 °C for 7 min. The amplified products were electrophoresed on a 1.5% agarose gel.

Statistical analysis

A statistical analysis was performed using the χ2 test. A value of P < 0.05 was considered statistically significant.

RESULTS
H. pylori clinical isolates

A total of 95 clinical H. pylori isolates were obtained from 40 male and 55 female patients with a mean age of 55.47 ± 16.87 years (age range of 17-89 years) and from three different ethnic groups (12 Malays, 59 Chinese and 24 Indians). The endoscopic findings were as follows: nonulcer dyspepsia (n = 76), gastric ulcer (n = 9), duodenal ulcer (n = 6) and both gastric and duodenal ulcers (n = 2). The OGDS findings were not available for two of the patients. Nonulcer dyspepsia was further classified as endoscopically normal stomach (n = 12), antrum-predominant gastritis (n = 43), corpus-predominant gastritis (n = 2) and pangastritis (n = 19). No significant association was found between patient ethnicity (Chinese versus non-Chinese) and the endoscopic findings (nonulcer dyspepsia versus peptic ulcer).

cagPAI genotype

Of the 95 H. pylori isolates, 87 (91.6%) were cagPAI-positive, and the remaining 8 (8.4%) lacked all selected cagPAI genes. For the cagPAI-positive strains, intact cagPAI genes were detected only in 3 isolates, whereas other isolates exhibited partially deleted (rearranged) cagPAI genes. Additionally, the IS605 gene was detected in 4 (4.2%) isolates. Further analyses indicated that the positivity of the H. pylori isolates was 61.1% (58/95), 53.7% (51/95), 4.2% (4/95), 82.1% (78/95), 76.8% (73/95) and 65.3% (62/95) for cag67, cag10, cag13, cagT, cagM and cagE, respectively.

As shown in Table 1, the presence of cagE and cagM in the cagI region was distributed evenly among the isolates from patients of different ethnicities, with no significant differences between the ethnicities. The cagT gene was frequently detected in isolates from the Malay patients, whereas cag13 was only detected in the isolates from the Chinese patients. The presence of cag67 was significantly higher in the isolates from the Chinese patients than in the isolates from the Malay patients and was lowest in the isolates from the Indian patients. The cagPAI genes were detected in greater than 50% of the isolates from the nonulcer and ulcer patients, except for cag13, which was only detected in the patients with a normal stomach or gastritis. Thus, different cagPAI genes were present in the majority of the isolates examined, irrespective of disease state.

Table 1 Distribution of Helicobacter pylori cagPAI genes isolated from patients of different ethnicities n (%).
cagPAI genesEthnic groupsP value
MalayChineseIndian
cagI region
cagE7 (58.3)41 (69.5)14 (58.3)0.541
cagM9 (75.0)45 (76.3)19 (79.2)0.948
cagII region
cagT11 (91.7)49 (83.1)18 (75.0)
cag1304 (6.8)0
cag106 (50.0)29 (49.2)16 (66.7)0.336
cag676 (50.0)47 (79.7)5 (20.8)< 0.0001

Of the 95 clinical isolates, only 3 (3.2%) contained an intact cagPAI, whereas 84 (88.4%) exhibited partial deletions within the cagPAI region, and 8 (8.4%) lacked the entire selected gene in the cagPAI. As shown in Table 2, thirteen isolates appeared to have a diverged cagPAI, in which the two selected genes for the cagI region had been deleted, although the genes in the cagII region were conserved, with at least one gene present. In contrast, ten strains had only one gene in either the cagI or the cagII region. The single gene detected was cagM, cagT and cag67 in 3, 6 and 1 isolate, respectively. The most commonly partially deleted cagPAI genotypes were detected in 26 (27.4%), 16 (16.8%) and 13 (13.7%) isolates, with cagPAI regions of types C, I and N, respectively. These isolates exhibited at least four genes in the cagPAI region, and the genes in the cagI region were conserved in these isolates. Additionally, the IS605 gene was detected in the isolates from the Chinese (n = 2) and Indian (n = 2) patients. Three of these isolates contained all of the selected genes for cagPAI, except cag13, and one isolate lacked the cag13 and cag10 genes.

Table 2 cagPAI genotypes of Helicobacter pylori clinical isolates n (%).
cagPAI typecagII regioncagI regionTotal
cag67cag10cag13cagTcagMcagE
A++++++3 (3.2)
B+-+---1 (1.1)
C++-+++26 (27.4)
D++-++-2 (2.1)
E++--++1 (1.1)
F++--+-1 (1.1)
G++----1 (1.1)
H+-----1 (1.1)
I+--+++16 (16.8)
J+--+-2 (2.1)
K+--+-+1 (1.1)
L+---++1 (1.1)
M+--+--2 (2.1)
N-+-+++13 (13.7)
O-+-++-2 (2.1)
P-+-+--2 (2.1)
Q---+++1 (1.1)
R---++-2 (2.1)
S---+--6 (6.3)
T----+-3 (3.2)
U------8 (8.4)

As shown in Table 3, three isolates with an intact cagPAI were obtained from the Chinese patients. Nineteen of the 59 (32.2%) isolates from the Chinese patients possessed partially deleted cagPAI regions of type C, compared with 25% and 16.7% of the isolates from the Malay and Indian patients, respectively. Interestingly, the isolates from the Indian patients showed a high proportion of partially deleted cagPAI regions of type N (37.5%) and deleted cagPAI regions (16.7%). An analysis of the disease state, with reference to strain genotype, revealed a relationship between the cagPAI genotype and disease severity. The isolates from those patients with peptic-ulcer disease (PUD) primarily exhibited cagPAI regions of type C, which contained more genes in the rearranged cagPAI genotypes. In contrast, a larger number of isolates from the patients with pangastritis or antrum-predominant gastritis possessed cagPAI regions of type I or N, respectively, compared with the isolates from the patients with peptic ulcers.

Table 3 Distribution of strains with intact, partially deleted (rearranged) or deleted cagPAI regions among patients of different ethnicities and disease states n (%).
cagPAI statusEthnic groupDisease state
MalayChineseIndianNormalGastritis (antral)PangastritisPUD
(n = 12)(n = 59)(n = 24)(n = 12)(n = 43)(n = 19)(n = 17)
Intact
Type A03 (5.1)01 (8.3)2 (4.7)00
Partially deleted
Type C3 (25.0)19 (32.2)4 (16.7)3 (25.0)9 (20.9)7 (36.8)7 (41.2)
Type I2 (16.7)14 (23.7)02 (16.7)5 (11.6)5 (26.3)3 (17.6)
Type N2 (16.7)2 (3.4)9 (37.5)1 (8.3)7 (16.3)2 (10.5)2 (11.8)
Deleted
Type U1 (8.3)3 (5.1)4 (16.7)1 (8.3)3 (7.0)1 (5.3)2 (11.8)
vacA genotype

The vacA s1m1, s1m2 and s2m2 genotypes were identified in 51.6% (49/95), 42.1% (40/95) and 4.2% (4/94) of the isolates, respectively. Additionally, two (2.1%) isolates possessed the vacA s1m1m2 genotype. The vacA s1m1 genotype was further classified as vacA s1am1 (63.3%, 31/49), s1cm1 (22.4%, 11/49) or s1as1cm1 (14.3%, 7/49), and the vacA s1m2 genotype was further classified as vacA s1am2 (65%, 26/40), s1cm2 (22.5%, 9/40) or s1as1cm2 (12.5%, 5/40). An analysis of the distribution of the vacA alleles with respect to patient ethnicity and disease state is shown in Table 4. We found a significant distribution of different vacA alleles according to patient ethnicity. The vacA s1m1 genotype was significantly more prevalent in the isolates from the Malay and Indian patients, whereas the vacA s1m2 genotype was predominantly detected in the isolates from the Chinese patients (P = 0.018). The vacA s2m2 genotype was only isolated from the Malay and Indian patients, and a mixed genotype of the m region was identified in the isolates from the Chinese patients. Further analyses of the vacA subgenotypes with respect to patient ethnicity revealed that the highest proportion of isolates harbouring the vacA s1am1 subgenotype were from Indian patients (93.8%, 15/16). The prevalence of the vacA s1cm1 genotype was similar in proportion in the isolates from the Malay (37.5%, 3/8) and Chinese (32%, 8/25) patients, although this subgenotype was not detected in the isolates from the Indian patients. In contrast, the vacA s1am2 subgenotype was detected in all of the ethnic groups, whereas the vacA s1cm2 subgenotype was only identified in the isolates from the Chinese patients. No significant difference was observed in the distribution of the vacA s1m1 and s1m2 strains with respect to the disease state (P = 0.686).

Table 4 Distribution of vacA alleles and metronidazole susceptibilities among Helicobacter pylori clinical isolates from patients of different ethnicities and disease states n (%).
cagPAI statusEthnic groupDisease state
MalayChineseIndianNormalGastritis (antral)PangastritisPUD
(n = 12)(n = 59)(n = 24)(n = 12)(n = 43)(n = 19)(n = 17)
Intact
Type03 (5.1)01 (8.3)2 (4.7)00
Partially deleted
Type C3 (25.0)19 (32.2)4 (16.7)3 (25.0)9 (20.9)7 (36.8)7 (41.2)
Type I2 (16.7)14 (23.7)02 (16.7)5 (11.6)5 (26.3)3 (17.6)
Type N2 (16.7)2 (3.4)9 (37.5)1 (8.3)7 (16.3)2 (10.5)2 (11.8)
Deleted
Type U1 (8.3)3 (5.1)4 (16.7)1 (8.3)3 (7.0)1 (5.3)2 (11.8)
Metronidazole susceptibility

In this study, metronidazole resistance was observed in 45.3% (43/95) of the isolates. The resistant strains exhibited MIC values ranging from 8 to > 256 mg/L, with an MIC value > 256 mg/L for the majority of the strains (62.8%, 27/43). As shown in Table 4, a strong association was observed between metronidazole susceptibility and patient ethnicity. The Chinese patients were more prone to infection with metronidazole-sensitive H. pylori strains, whereas the Malay and Indian patients were more likely to be infected with the antibiotic-resistant strains. The strains isolated from the patients with PUD were more sensitive to metronidazole than the strains from the patients with pangastritis, although this difference was not statistically significant. DNA sequence analyses of the rdxA gene revealed the presence of missense, frameshift and nonsense mutations in 30, 3 and 4 isolates, respectively. All of the tested metronidazole-sensitive strains harboured only missense mutations, whereas all three types of mutations were identified in the antibiotic-resistant strains.

Distribution of H. pylori cagPAI genotypes, vacA alleles and metronidazole susceptibilities

As shown in Table 5, the isolates with intact cagPAI regions carrying the vacA s1m2 allele were sensitive to metronidazole. Of the isolates with a rearranged cagPAI region, the type C strains predominantly harboured the vacA s1m2 allele, whereas a high proportion of the types I and N cagPAI strains harboured the vacA s1m1 allele. However, these strains were found to be more sensitive to metronidazole. In contrast, the isolates exhibiting a deleted cagPAI region predominantly carried the vacA s1m2 allele and were more resistant to metronidazole.

Table 5 Distribution of Helicobacter pylori cagPAI genotypes, vacA alleles and metronidazole susceptibilities n (%).
cagPAI statusvacA
Metronidazole susceptibility
vacA/metronidazole susceptibility
s1m1s1m2SensitiveResistants1m1/sensitives1m1/resistants1m2/sensitives1m2/resistant
Intact (n = 2)102 (100)2 (100)0002 (100)0
Partially deleted
Type C (n = 26)10 (38.5)16 (61.5)14 (53.8)12 (46.2)5 (19.2)5 (19.2)9 (34.6)7 (26.9)
Type I (n = 16)12 (75.0)4 (25.0)10 (62.5)6 (37.5)7 (43.8)5 (31.2)3 (18.8)1 (6.2)
Type N (n = 13)11 (84.6)2 (15.4)8 (61.5)5 (38.5)7 (53.8)4 (50.0)1 (12.5)1 (12.5)
Deleted (n = 6)21 (16.7)5 (83.3)3 (37.5)5 (62.5)01 (16.7)2 (33.3)3 (50.0)
DISCUSSION

The cagPAI and vacA genotypes are widely used to characterise the virulence of H. pylori and the relationship of such virulence to disease severity, although direct associations with peptic ulcers and gastric cancer have not been established[22,23]. Rather, the development of severe histological changes in the gastric mucosa may depend on the synergistic effect of bacterial and host factors[24].

The genotypic characteristics of the H. pylori cagPAI genes show great variability worldwide. The rearrangement of the cagPAI varies between different populations and geographical regions, with variations of 50%-65% in areas of the Indian subcontinent[25,26], 32% in France[27], 9% in Sweden[28] and the United States[29] and 1% in Japan[30]. In the present study, more than 90% of the clinical isolates were positive for at least one of the selected cagPAI genes. The cagPAI genes tested in this study did not include the cagA gene, as we previously demonstrated[31] that the majority of the H. pylori strains circulating in our study population were cagA-positive. In the six genetic loci studied, the most frequently deleted gene was cag13, and the least frequently deleted gene was cagT. The IS605 insertion sequence was present in less than 5% of the H. pylori isolates examined, and the majority of the isolates harboured a rearranged cagPAI gene. Analyses of the individual cagPAI genes showed that cag67 was more conserved in the isolates from the Chinese patients, whereas the rearrangement of cagE occurred at a higher frequency in the isolates from the Malay and Indian patients, although this difference was not significant.

We also observed that the majority of the H. pylori clinical isolates expressed partially deleted cagPAI regions and that only few isolates exhibited either intact or deleted cagPAI regions. Previous studies have reported the occurrence of different proportions of intact cagPAI in their isolates. Ali et al[32] found that 37.4% of isolates exhibited intact cagPAI regions, whereas other researchers[30,33] reported that more than 96% of isolates contained intact cagPAI. In the present study, the isolates expressing intermediate genotypes (deletions within the cagPAI region) were more commonly encountered than those isolates with intact or deleted cagPAI regions. Moreover, a large proportion of the isolates exhibiting cagPAI rearrangement lacked an IS605 sequence, suggesting that this insertion sequence did not play a role in cagPAI disruption in the clinical isolates.

Additionally, we found that no single gene in the cagPAI region can be used as a genetic marker for an intact cagPAI because a large proportion of the clinical isolates exhibited gene rearrangement in the cagPAI region. Due to the region’s large size (approximately 40 kbp), the absence of these genes in certain clinical isolates did not necessarily indicate the complete absence of the cagPAI, as indicated in 9 isolates positive for a single gene. Regarding cagPAI rearrangement, 19 different genotypes (types B to T) have been identified. The three most common cagPAI genotypes identified in the clinical strains revealed that the genes at the left end of the cagPAI region were more unstable and prone to rearrangement than the genes in the middle and at the right end of the cagPAI region. This finding was supported by analyses of the individual genes, which demonstrated more gene rearrangement at the left end of the cagPAI region (cag13, cag10 and cag67). Therefore, the genes in the middle and at the right end of the cagPAI region may play an important role in the pathogenesis of H. pylori infection in the study population.

In the current study, specific cagPAI genotypes were isolated from patients of different ethnicities. All of the isolates with intact cagPAI regions and less cagPAI rearrangement were from the Chinese patients, whereas the Indian patients were infected with strains exhibiting more rearranged or deleted cagPAI regions, and the Malay patients tended to be infected with various cagPAI genotypes. Thus, the presence of different cagPAI genotypes in different ethnicities may be related to the genetic characteristics of both the colonising H. pylori strain and the host. Although the relationship between the cagPAI genotype and disease state was unclear, we observed that certain types of infecting strains exhibited different cagPAI rearrangements in nonulcer and ulcer patients.

A significant difference was observed in the prevalence of vacA genotypes among patients of distinct ethnicities. As the Chinese patients were more likely to be infected with the vacA s1m2 strains, these strains may be regarded as more pathogenic, consistent with previous reports showing a higher frequency of peptic ulcers and gastric cancer in the Chinese patients in a similar study population[15,34]. In our study population, the smaller proportion of patients with PUD than NUD may have contributed to the lack of a significant association between the endoscopic findings and patient ethnicity, as analysis was conducted on the samples with positive cultures. Additionally, we demonstrated that the vacA s1m2 genotype was significantly associated with enhanced gastric inflammation[10]. In contrast, the vacA s2m2 strains were isolated only from the Malay and Indian patients, suggesting that these strains are less virulent. Consistent with this finding, Chinese ethnicity has been associated with infection by strains lacking the vacA s2m2 allele[35,36]. As Miernyk et al[37] recently reported a high proportion of vacA s2m2 strains isolated from Alaskan natives, it would be interesting to examine whether the disease outcome in Alaskan natives is similar to the outcome in the Malay and Indian patients in our study population. The current study also revealed the proportions of different vacA subgenotypes in each ethnic population. More specifically, the vacA s1cm1 genotype was not detected in the isolates from the Indian patients, whereas the vacA s1cm2 genotype was only detected in the isolates from the Chinese patients.

A previous report demonstrated a higher prevalence of resistance to metronidazole than to other antibiotics in H. pylori isolates from Malaysia[14]. In the current study, we attempted to further relate the metronidazole susceptibility of H. pylori to patient ethnicity and to correlate this susceptibility with the cagPAI and vacA genotypes. We noted a significant association between the metronidazole-sensitive H. pylori strains and Chinese ethnicity, whereas the Malay and Indian patients were more likely to be infected with the metronidazole-resistant strains. This finding may reflect differences in metronidazole use between different ethnic populations. The pattern of metronidazole susceptibility in different ethnicities paralleled the specific genotypes of the infecting strains. For example, the strains of the specific cagPAI genotype and vacA allele isolated from the Chinese patients were more sensitive to metronidazole.

The presence of frameshift and nonsense mutations in the antibiotic-resistant H. pylori strains suggested that these mutations confer resistance to metronidazole. However, more than half of the resistant strains exhibited missense mutations that were also detected in the antibiotic-sensitive strains. This finding implied that rdxA is not the only gene involved in the metronidazole-resistant phenotype and thus is not a reliable epidemiological marker. Rather, other genes or mechanisms may be implicated in the generation of resistance[38-40].

We observed no association between the specific H. pylori genotypes and the strains’ antibiotic susceptibilities in severe disease. These results may be complicated by the fact that most of the patients in our study population had gastritis. Although the cagPAI and vacA alleles are important virulence factors in infection, the development of disease is likely to involve a highly complex interplay of many bacterial and/or host factors.

The distribution of the vacA s1m2 genotype was broad, as this genotype was detected in the majority of the strains with intact cagPAI regions or cagPAI rearrangement of type C (positive for more cagPAI genes than other types) and the metronidazole-resistant strains. This finding may indicate that the vacA s1m2 strains have variable pathogenic properties when combined with other genotypic characteristics. The isolates with the deleted cagPAI regions were also primarily linked to metronidazole resistance. As these isolates may induce less inflammation in the host gastric epithelia, their genotypic characteristics may reduce antibiotic delivery and thus hinder the eradication of H. pylori. In contrast, the vacA s1m2 genotype could not contribute to metronidazole resistance, as this genotype was distributed evenly between the antibiotic-sensitive and -resistant strains[41,42].

In conclusion, we report a large proportion of H. pylori isolates harbouring cagPAI rearrangement and demonstrate that metronidazole susceptibility varies with patient ethnicity. The distinct distribution of the H. pylori cagPAI genotypes, vacA alleles and metronidazole susceptibilities in the different ethnicities of Malaysia may contribute to varying risk of gastroduodenal diseases. These distinct, patient ethnicity-associated H. pylori genotypes may have important clinical and epidemiological implications. Finally, the present study of H. pylori-specific genotypes from different host genetic backgrounds enhances our understanding of bacterium-host interactions and bacterial ecology in various niches. Further information on the characteristics of H. pylori will allow a more precise identification of virulent strains and a better definition of risk factors in susceptible hosts.

ACKNOWLEDGMENTS

We thank Universiti Kebangsaan Malaysia for providing both the permission and the facilities to conduct and publish this research. We are also grateful to all of the staff of the Endoscopy and Histopathology Units at UKMMC for their technical help.

COMMENTS
Background

The prevalence of Helicobacter pylori (H. pylori) infection in Malaysia consistently revealed ethnic differences, with Indians exhibiting higher infection rates than the Chinese and Malays. In contrast, peptic-ulcer disease and gastric carcinoma are known to be more prevalent in the Chinese and lowest in the Indians. The great genetic diversity of H. pylori may play an important role in the consequences of infection in different hosts. The cagPAI and vacA genes are well-established H. pylori virulence factors that interact with the host cells and disrupt downstream signalling pathways. The cagPAI structures, vacA alleles and metronidazole susceptibilities of different strains in hosts of varying genetic makeup may enhance the knowledge of bacterium-host interactions in H. pylori infections in multiethnic populations.

Research frontiers

The specific genotypes stemming from the cagPAI and vacA alleles were identified in H. pylori strains isolated from different ethnic groups. The strains isolated from the Chinese and peptic-ulcer disease (PUD) patients were more sensitive to antibiotics, indicating a selective advantage that occurred during early infection and persisted in chronic infection. In the present study, however, no association between the specific H. pylori genotypes and PUD could be determined due to the small number of PUD cases. Consistent with this observation, previous epidemiological studies have reported that less than 20% of patients infected with H. pylori are diagnosed as PUD.

Innovations and breakthroughs

Past reports have highlighted the importance of H. pylori genetic diversity in chronic infection. The current study emphasises and adds to findings from the same institution, demonstrating that the genetic background of the H. pylori strains may play an important role in the risk of gastroduodenal disease in different ethnic groups.

Applications

The results of this study provide insight into the effects of the genomic diversity of H. pylori on hosts of different genetic backgrounds. Therefore, these findings can be used in the development of advanced screening tools for diagnosing and determining the prognosis of H. pylori infection.

Terminology

The term cagPAI is defined as the cag pathogenicity island, a common gene sequence believed to be responsible for pathogenesis. This sequence contains approximately 40 kbp of nucleotides encoding over 40 genes. The pathogenicity island is typically absent from the H. pylori strains isolated from human carriers of H. pylori who remain asymptomatic.

Peer review

The authors investigated the diversity of the H. pylori virulence factors (the cagPAI and vacA alleles) and metronidazole susceptibilities of strains isolated from patients of different ethnicities. The association between specific H. pylori genotypes and patient ethnicity provides insight into the pathogenesis of H. pylori infection in different hosts and possibly the different risk factors in H. pylori infection.

Footnotes

P- Reviewer Mori N S- Editor Gou SX L- Editor A E- Editor Zhang DN

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