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Introduction

Helicobacter pylori (H. pylori) is a Gram-negative, spiral-shaped, microaerophilic bacterium that colonizes the human gastric mucosa (1). H. pylori infection can cause chronic gastritis, peptic ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue lymphoma (2–4). More than half of the world's population is infected with H. pylori (5). However, the majority of infected individuals remain asymptomatic. There is an interplay between host genetic susceptibility, environmental factors and bacterial virulence factors, which influences the outcome of H. pylori-associated diseases (6–8). Polymorphisms of several virulence genes, including cytotoxin-associated gene A (cagA), vacuolating toxin A (vacA), induced by contact with epithelium gene A (iceA), blood group antigen binding adhesin and duodenal ulcer promoting gene A are considered to increase the risk for the development of upper gastrointestinal diseases (9). Among these genes, cagA and vacA have been investigated extensively.

CagA is an oncogenic protein, and is a major virulence factor associated with gastric cancer (10,11). CagA is injected into host epithelial cells via the type IV secretion system encoded by cag pathogenicity island (cagPAI) following H. pylori infection (12). CagA undergoes phosphorylation at its EPIYA motif by the Abl and Src family tyrosine kinases (13). Once tyrosine is phosphorylated, the cagA EPIYA motifs serve as a recognition site for Src homology phosphotyrosine phosphatase 2 (SHP2), and activate an intracellular signal transduction pathway that leads to cytoskeletal rearrangement and cell elongation, known as the ‘hummingbird’ phenotype (14). This increases the risk of developing precancerous lesions (15).

The EPIYA motif is located within the C-terminal of cagA, and is formed of the conserved amino-acid residues Glu-Pro-Ile-Tyr-Ala. EPIYA can be further classified into four types of motif: EPIYA-A, -B, -C and -D, based on the flanking amino acid sequences (16). H. pylori is classified as East Asian-type cagA and western-type cagA, according to the composition of the EPIYA-A, -B, -C and D motifs. The western-type cagA is mainly cagA-ABC, whereas the East Asian-type is mainly cagA-ABD (17). The EPIYA-C and EPIYA-D motifs act as phosphorylation sites for SHP-2 (18). The EPIYA-D segment exhibits a greater degree of tyrosine phosphorylation and higher binding affinity to SHP2 than the EPIYA-C segment and shows higher virulence (2). East Asian-type cagA is associated with a higher risk of peptic ulcers or gastric cancer within the same geographical area compared with western-type cagA (19). Epidemiological surveys show that ~50–60% of H. pylori strains in western countries contain the cagA gene, which increases the risk of peptic ulcers and gastric cancer (20). Although 90–100% of H. pylori strains in East Asian countries are cagA-positive, cagA-positive strains may not be associated with clinical outcomes (21).

VacA is a pore-forming toxin, which has several effects on epithelial cells. In addition to inducing vacuolation (22), vacA can induce membrane channel formation, which leads to the release of cytochrome c from mitochondria and results in apoptosis (23). Notably, it has immunomodulatory effects through inhibiting T-cell activation and proliferation (6,19). There are four sequence diversity regions of vacA closely associated with H. pylori vacuolating activity, namely signal region (s-), deletion region (d-), intermediate region (i-) and middle region (m-) (4). The cytotoxicity of vacA is determined by variability in the structure of the vacA gene (24). The s- and m- regions of vacA are the two main polymorphic regions and serve as markers of H. pylori virulence and the risk of associated diseases (25).

Several studies have shown that iceA has two main allelic variants, namely iceA1 and iceA2 (26). The expression of iceA1 is upregulated upon contact between H. pylori and human epithelial cells. The iceA1 genotype is linked with enhanced mucosa interleukin-8 expression and acute inflammation (27). Epidemiological data shows that the geographical distribution of iceA genotypes varies. The iceA1 strains mainly occur in Japan and South Korea, whereas iceA2 strains are predominant in the United States and Columbia (28). A previous meta-analysis showed that the prevalence of iceA1 was significantly higher in East Asian countries than in western countries, whereas the prevalence of iceA2 was higher in western countries than in East Asian countries (26). Functionally, the iceA1 genotype is associated with peptic ulcers (29) and the iceA2 genotype with the occurrence of chronic gastritis (28). To date, the majority of studies have shown that the iceA gene is another virulence gene independent of cagA and vacA (30).

Guizhou, a province located in southwest China, is a multi-ethnic society. In particular, those in Qiannan, Buyei and Miao Autonomous Prefecture have a particular lifestyle and are different from other ethnic groups. The individuals living here often use herbal medicine to treat diseases, including stomach disorders. As Xie et al (31) reported, such traditional medicine may have an effect on bacteria in the stomach, including H. pylori. Although investigations on the role of H. pylori virulence cagA and vacA genotypes have been performed worldwide, the associations between H. pylori virulence genotype and gastroduodenal diseases, ethnicity or economic conditions in Guizhou province remain to be fully elucidated. Therefore, the present study aimed to investigate these associations, which may facilitate diagnosis and therapeutic strategies for gastroduodenal diseases.

Materials and methods

Clinical samples

Gastric pylorus mucosa biopsy samples were obtained by endoscopy from patients with gastric disorders at the First Affiliated Hospital of Guizhou Medical University (Guizhou, China) and the People's Hospital of Qiannan Autonomous Prefecture (Duyun, China) between January and December 2016. Clinicopathological data were collected, and written informed consent was obtained from all patients. Patients with the following conditions were excluded from the present study: A tendency to bleed, lactating or pregnant women, the inability to undergo surgery to the UGI tract, and severe cardiovascular or hepatic disease. All protocols were approved by the Ethical Committee of the First Affiliated hospital of Guizhou Medical University. All procedures contributing to the study complied with the Declaration of Helsinki.

H. pylori isolation and bacterial DNA extract

Gastric mucosa samples were cut into small sections, homogenized and smeared on the surface of Brain Heart Infusion agar with 10% sheep blood (Qingdao Hope Biol-Technology Co., Ltd., Qingdao, China) and antibiotic supplement (H. pylori selective supplement, Thermo Fisher Scientific Oxoid, Ltd., Basingstoke, UK). The plates were incubated at 37°C for 3–5 days under microaerophilic conditions. H. pylori was identified through colonial morphology, Gram staining, a urease test, and H. pylori-specific 16S rRNA gene fragment polymerase chain reaction (PCR) amplification. The colony of H. pylori was smooth and translucent, and the morphology was Gram-negative with spiral-shaped bacilli. The confirmed colonies were subcultured to single colonies on fresh medium. Following incubation at 37°C for 3–5 days, the colonies were subjected to DNA extraction using an Ezup column bacteria genomic DNA purification kit (Sangon Biotech Co., Ltd., Shanghai, China), according to the manufacturer's protocol.

PCR amplification and sequencing

PCR assays to amplify cagA and sequence its C-terminal region were performed according to the report by Sicinschi et al (14). Primers (Sangon Biotech Co., Ltd., Shanghai, China) used for amplification of the cagA, vacA and iceA genes in the present study are shown in Table I. Following DNA extraction from pure culture of H. Pylori isolates as previously mentioned, PCR assays were performed in a volume of 26 µl containing 1 µl forward primer, 1 µl reverse primer, 4 µl genomic DNA, 13 µl 2X Taq PCR Master Mix (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) and 7 µl ddH2O. Table I summarizes the expected size of the PCR products and cycling conditions for cagA gene, vacA subtype gene and iceA allelic genes (14,32–34). All runs included one negative (ddH2O) and one positive (NCTC 11637 or H. pylori 26695) DNA control, and DNA ladder markers (Tiangen Biotech Co., Ltd., Beijing, China). A total of 6 µl of amplified PCR products was then resolved by electrophoresis on 2% agarose gels run in acetate EDTA buffer, and stained with ethidium bromide. The PCR product was visualized under ultraviolet light. The cagA C-terminal PCR products were sent to Sangon Biotech Co., Ltd. for Sanger sequencing. The gene sequences were translated into amino acid sequences using Bioedit software (version 7.1.3.0; http://www.bioedit.com/). Phylogenetic tree cluster analysis of cagA carboxyl terminal variable region was constructed using MEGA software (version 6.0; http://www.megasoftware.net/megamac.php), based on neighbor joining. The control strain NCTC 11637 was obtained from the State Key Laboratory of Infectious Disease Prevention and Control (National Institute for Communicable Disease Control and Prevention, Beijing, China), and the amino acid sequences of H. pylori 26695 were obtained from GenBank (https://www.ncbi.nlm.nih.gov/nuccore/CP003904.1).

Table I. Primer sequences and reaction conditions for polymerase chain reaction.

Table I.

Primer sequences and reaction conditions for polymerase chain reaction.

Primer	Gene	Primer sequence	Size (bp)	Amplification condition
16S rRNA	16S rRNA-F	5′-CTTGCTAGAGTGCTGATTA-3′	550	35 cycles: 94°C for 30 sec; 55°C for 30 sec; 72°C for 30 sec
	16S rRNA-R	5′-TCCCACACTCTAGAATAGT-3′
cagA 5′-end conserved region	cagA F	5′-GATAACAGGCAAGCTTTTGAGG-3′	349	30 cycles: 94°C for 1 min; 55°C for 1 min; 72°C for 1 min
	cagA R	5′-CTGCAAAAGATTGTTTGGCAGA-3′
cagA 3′-end variable region	cagA-VF	5′-ACCCTAGTCGGTAATGGGTTA-3′	591–856	30 cycles: 94°C for 1 min; 50°C for 1 min; 72°C for 1 min
	cagA-VR	5′-GTAATTGTCTAGTTTCGC-3′
cagPAI empty site	Empty site-F	5′-ACATTTTGGCTAAATAAACGCTG-3′	535	30 cycles: 94°C for 1 min; 55°C for 1 min; 72°C for 1 min
	Empty site-R	5′-GGTTGCACGCATTTTCCCTTAATC-3′
iceA1	iceA1-F	5′-GCTTGTAACGATAAGAAACGCCAGAT-3′	297	35 cycles: 94°C for 30 sec; 55°C for 30 sec; 72°C for 30 sec
	iceA1-R	5′-GGAATGAGCTTGTATTTAGAGCCGAT-3′
iceA2	iceA2-F	5′-GTTGGGTATATCACAATTTAT-3′	229/334	30 cycles: 94°C for 30 sec; 52°C for 30 sec; 72°C for 45 sec
	iceA2-R	5′-TTRCCCTATTTTCTAGTAGGT-3′
vacA-s1a	vacA-s1a-F	5′-CTCTCGCTTTAGTAGGAGC-3′	213	30 cycles: 94°C for 30 sec; 60°C for 30 sec; 72°C for 45 sec
	vacA-s1a-R	5′-CTGCTTGAATGCGCCAAAC-3′
vacA-s1b	vacA-s1b-F	5′-AGCGCCATACCGCAAGAG-3′	187
	vacA-s1b-R	5′-CTGCTTGAATGCGCCAAAC-3′
vacA-s1c	vacA-s1c-F	5′-CTCTCGCTTTAGTGGGGYT-3′	213
	vacA-s1c-R	5′-CTGCTTGAATGCGCCAAAC-3′
vacA-s2	vacA-s2-F	5′-GCTAACACGCCAAATGATCC-3′	199
	vacA-s2-R	5′-CTGCTTGAATGCGCCAAAC-3′
vacA-m1a	vacA-m1a-F	5′-GGTCAAAATGCGGTCATGG-3′	290
	vacA-m1a-R	5′-CCATTGGTACCTGTAGAAAC-3′
vacA-m1b	vacA-m1b-F	5′-GGCCCCAATGCAGTCATGGAT-3′	291
	vacA-m1b-R	5′-GCTGTTAGTGCCTAAAGAAGCAT-3′
vacA-m2	vacA-m2-F	5′-GGAGCCCCAGGAAACATTG-3′	352
	vacA-m2-R	5′-CATAACTAGCGCCTTGCAC-3′

[i] cagA, cytotoxin associated gene A; iceA induced by contact with epithelium gene A; vacA, vacuolating cytotoxin A; F, forward; R, reverse.

Statistical analysis

All data were analysed using SPSS 19.0 (IBM SPSS, Armonk, NY, USA), χ2 test and Fisher's exact test were used for the analysis of categorical data. P<0.05 was considered to indicate a statistically significant difference.

Results

Clinical and pathological information

A total of 73 H. pylori strains were isolated from patients with upper gastroduodenal disorders. The demographics of these patients are shown in Table II. Among the 73 cases, 41 were men with a mean age of 38.85±19.43 years, and range of 3–80 years, and 32 were women with a mean age of 39.94±18.93 years, and range of 4–66. A total of 60 strains were isolated from Han groups, and 13 from other minority ethnic groups, including Miao, Dong, Tujia, Buyi, Bai and Yi groups. A total of 60 strains were isolated from Guiyang city, and 13 from Qiannan autonomous prefecture. A total of 31 cases were from urban populations, and 42 were from suburban populations. Finally, 54 strains were isolated from patients with gastritis and 19 from patients with peptic ulcers, the latter comprising eight with gastric ulcer and 11 with duodenal ulcer. PCR products of 550 bp represented H. pylori-specific 16S rRNA, as shown in Fig. 1A.

Figure 1. PCR amplification of H. pylori cagA, vacA and iceA genes. (A) Gel electrophoresis of genus-specific 16S rRNA PCR products from H. pylori isolates. M, Marker II; 1–14, positive sample for 16Sr RNA PCR; 15, negative control; 16, positive control (NCTC 11637). (B) Gel electrophoresis of clinical samples with different cagA N-terminals. M, 100 bp DNA ladder marker; 1–20, clinical sample; 23, negative control; 24, positive control (NCTC 11637). (C) Gel electrophoresis of clinical samples with different cagA EPIYA patterns. M, 100 bp DNA ladder marker; 1–22, clinical sample; 23, negative control; 24, positive control (NCTC 11637). (D) Gel electrophoresis of H. pylori genotyping of vacA alleles. M, 100 bp DNA ladder marker; s1a, s1b, s1c and s2, vacA signal region; m1a, m1b and m2, vacA middle region. (E) Gel electrophoresis of H. pylori genotyping of iceA1 alleles. M, 100 bp DNA ladder marker; 1–11, clinical sample; 12, negative control; 13, positive control (H. pylori 26695). (F) Gel electrophoresis of H. pylori genotyping of iceA2 alleles. M, 100 bp DNA ladder marker; 1–5 and 8–9, negative clinical sample; 6–7, positive clinical sample (iceA2-229 bp); 10, negative control; 11, positive control (iceA2-334 bp). H. pylori, Helicobacter pylori; cagA, cytotoxin associated gene A; vacA, vacuolating cytotoxin A; iceA induced by contact with epithelium gene A; PCR, polymerase chain reaction.

Table II. Demographic characteristics of patients.

Table II.

Demographic characteristics of patients.

Characteristic	Patients (n)	Age (years)a	Range
Sex
Male	41	38.85±19.43	3–80
Female	32	39.94±18.93	4–66
Ethnicity
Han	60	40.68±19.64	3–80
Ethnic minority
Miao	4	25.75±19.45	7–44
Dong	2	23±22.63	7–39
Tujia	2	36.5±16.26	25–48
Buyi	2	42.5±0.71	42–43
Bai	2	36.5±2.12	35–38
Yi	1	50	–
Place of residence
Guiyang city	60	37.97±20.73	3–80
Qiannan autonomous prefecture	13	45.62±4.68	38–53
Urban	31	42.19±21.19	3–80
Suburban	42	36.41±19.49	4–72
Clinical disease
Gastritis	54	38.11±20.34	3–80
Peptic ulcer	19	42.79±14.90	9–60

a Age is expressed as the mean ± standard deviation.

Polymorphism of cagA

The cagA N-terminal conserved region (cagA 5′-end) and C-terminal variable region (cagA 3′-end) were amplified by PCR using the specific primers (Table I). PCR products of 349 bp represented the 5′-end fragments, as shown in Fig. 1B, and were present in all H. pylori isolates (n=73, 100%). CagPAI empty site PCR produced negative results in all of these strains, indicating the absence of cagA-negative strains in the mixture. PCR products within the range of 450–650 bp represented the 3′-end fragments, as shown in Fig. 1C. All H. pylori isolates were found to contain the 3′-end variable region expressing various EPIYA motifs. Notably, four distinct PCR products (500, 600, 610 and 550 bp amplicons) were obtained from the various H. pylori isolates (Fig. 1C).

Polymorphism of vacA

The vacA gene polymorphism was also analyzed. The vacA gene has variation regions including signal (s-) and middle (m-) regions (24). Usually, alleles are further divided into sub-alleles, including s1a, s1b, s1c, s2, m1a, m1b and m2 (34). The H. pylori isolates were screened for all sub-alleles by the PCR assay, as shown in Fig. 1D. vacA s1c/m1b and s1c/m2 genotypes were identified in 24.66 and 65.75%, respectively, whereas the s1c/m1b/m2 mixed genotype was only identified in 9.59%, as shown in Table III. The distribution of vacA genotypes in Guizhou province is shown in Table III. No significant association between the vacA genotypes and clinical outcomes was identified (P=1.000). There was also no significant association between the vacA genotypes and age, place of residence (Guiyang city vs. Qiannan autonomous prefecture, urban vs. suburban), or ethnic group (P=0.605, P=0.400, P=0.718 and P=0.210, respectively), as shown in Tables IV and V.

Table III. Distribution of cagA genotypes in Guizhou province.

Table III.

Distribution of cagA genotypes in Guizhou province.

Genotype	n (%)	Guiyang city strains (n)	Qiannan autonomous prefecture strains (n)
Western-type cagA-AB	5 (6.85)	5	0
Western-type cagA-ABC	3 (4.11)	3	0
East Asia-type cagA-ABD	63 (86.30)	50	13
East Asia-type cagA-BD	2 (2.74)	2	0
vacA s1c/m1b	18 (24.66)	13	5
vacA s1c/m2	48 (65.75)	41	7
vacA s1c/m1b/m2	7 (9.59)	6	1
iceA1	58 (79.45)	49	9
iceA2	2 (2.74)	1	1
iceA1+iceA2	13 (17.81)	10	3

[i] cagA, cytotoxin associated gene A; vacA, vacuolating cytotoxin A; iceA, induced by contact with epithelium gene A.

Table IV. Distribution of virulence genotypes by clinical disease and age of patients.

Table IV.

Distribution of virulence genotypes by clinical disease and age of patients.

Genotype	Gastritis (n=54)	Peptic ulcer (n=19)	P-value	Age <18 years (n=16)	Age >18 years (n=57)	P-value
cagA-AB	5	0	0.417	0	4	0.180
cagA-ABC	3	0		2	1
cagA-ABD	44	19		14	50
cagA-BD	2	0		0	2
vacA s1c/m1b	13	5	1.000	5	13	0.605
vacA s1c/m2	36	12		9	39
vacA s1c/m1b/m2	5	2		2	5
iceA1	41	17	0.146	12	46	0.679
iceA2	1	1		0	2
iceA1+iceA2	12	1		4	9

[i] cagA, cytotoxin associated gene A; vacA, vacuolating cytotoxin A; iceA, induced by contact with epithelium gene A.

Table V. Distribution of cagA genotypes by place of residence and ethnicity of patients.

Table V.

Distribution of cagA genotypes by place of residence and ethnicity of patients.

	Place of residence						Ethnic group
Genotype	Guiyang city	Qiannan autonomous prefecture	P-value	Urban	Suburban	P-value	Han	Minority	P-value
CagA-AB	5	0	0.845	3	2	0.602	4	1	1.000
CagA-ABC	3	0		2	1		3	0
cagA-ABD	50	13		25	38		51	12
CagA-BD	2	0		1	1		2	0
vacA s1c/m1b	13	5	0.400	7	11	0.718	13	5	0.210
vacA s1c/m2	41	7		22	26		42	6
vacA s1c/m1b/m2	6	1		2	5		5	2
iceA1	49	9	0.304	24	34	0.884	46	12	0.621
iceA2	1	1		1	1		2	0
iceA1/iceA2	10	3		6	7		12	1

[i] cagA, cytotoxin associated gene A; vacA, vacuolating cytotoxin A; iceA, induced by contact with epithelium gene A.

Polymorphism of iceA

The polymorphism of iceA genes was also assessed. Gel electrophoresis genotyping of H. pylori iceA1 and iceA2 alleles is shown in Fig. 1E and F, respectively. Among the 73 cases, the iceA1 and iceA2 genotypes were present in 79.45% (58/73) and 2.74% (2/73), respectively. The iceA1/iceA2 mixed genotype occurred in 17.81% (13/73) of cases. The distribution of iceA genotypes in Guizhou province is shown in Table III. No significant association was found between iceA genotypes and disease outcomes, age, place of residence (Guiyang city, vs. Qiannan autonomous prefecture, urban vs. suburban), or ethnic group (P=0.146, P=0.679, P=0.304, P=0.884 and P=0.621, respectively), as shown in Tables IV and V.

EPIYA motifs of cagA

As described above, the EPIYA motif patterns were determined for each strain. A comparison of the results indicated that the EPIYA genotypes of the 500, 600, 610 and 550 bp amplicons were these of the cagA-AB, -ABC, -ABD and -BD genotypes, respectively. The structural polymorphism of the cagA amino acid sequence is shown in Fig. 2. It was noted that cagA EPIYA-ABD (63/73, 86.30%) was the predominant genotype in the present study, followed by cagA-AB (5/73, 6.85%), cagA-ABC (3/73, 4.11%) and cagA-BD (2/73, 2.74%). The distribution of cagA genotypes in Guizhou province is shown in Table III. However, statistical analysis revealed no significant correlation between the disease outcome and the cagA genotypes (P=0.417). There was also no significant correlation between the cagA genotypes and age, place of residence (Guiyang city, vs. Qiannan autonomous prefecture, urban vs. suburban), or ethnic group (P=0.180, P=0.845, P=0.602 and P=1.000, respectively), as shown in Tables IV and V.

Figure 2. Structural polymorphism in the cagA amino acid sequence of East Asian-type and western-type cagA. cagA, cytotoxin associated gene A.

The results of phylogenetic tree cluster analysis of the cagA carboxyl terminal variable region is shown in Fig. 3. There was a clustering association between western-type cagA-AB and cagA-ABC; there was also a clustering association between East Asian-type cagA-ABD and cagA-BD. All five strains with western-type cagA-AB and three strains with western-type cagA-ABC were isolated from patients with chronic gastritis; 19 strains with East Asian-type cagA-ABD were isolated from patients with peptic ulcers, and a further 46 with East Asia-type cagA were isolated from patients with chronic gastritis. As mentioned above, it appeared that East Asia-type cagA strains may exhibit higher virulence than western-type cagA strains.

Figure 3. Phylogenetic tree cluster analysis of the cagA carboxyl terminal variable region. On the inner side of the phylogenetic tree, red branches are western-type cagA-ABC (indicated in circle 1); purple branches are western-type cagA-AB (circle 1); blue branches are East Asia-type cagA-ABD; cyan branches are East Asia-type cagA-BD (circle 2). On the outer side of the phylogenetic tree, green circles are gastritis; orange circles are for duodenal ulcer; blue circles are for gastric ulcer; black circles are for compound ulcer, including duodenal ulcer and gastric ulcer. cagA, cytotoxin associated gene A.

The frequencies of the four EPIYA or EPIYA-like motifs are shown in Table VI. In total, 212 EPIYA motifs were obtained from 73 cagA sequences. The most frequent EPIYA motif was EPIYA (197/212, 92.92%), followed by EPIYT (8/212, 3.77%), ESIYA (6/212, 2.83%) and ESIYT (1/212, 0.47%). The EPIYA-B motif had a high degree of variation in the five amino acids (EPIYA, EPIYT, ESIYA or ESIYT). In addition, 15 EPIYA-like motifs (EPIYT, ESIYA, ESIYT) were isolated from patients with gastritis.

Table VI. Frequencies of EPIYA motifs.

Table VI.

Frequencies of EPIYA motifs.

Motif type	EPIYA	EPIYT	ESIYA	ESIYT	Total
All cagA type
All motifs	197	8	6	1	212
A motifs	71	0	0	0
B motifs	59	7	6	1
C motifs	2	1	0	0
D motifs	65	0	0	0
Western-type-ABC
All motifs	5	4	0	0	9
A motifs	3	0	0	0
B motifs	0	3	0	0
C motifs	2	1	0	0
Eastern-type-ABD
All motifs	181	2	5	1	189
A motifs	63	0	0	0
B motifs	55	2	5	1
D motifs	63	0	0	0
Western-type-AB
All motifs	7	2	1		10
A motifs	5	0	0
B motifs	2	2	1
Eastern-type-BD
All motifs	4	0	0		4
B motifs	2	0	0
D motifs	2	0	0

[i] cagA, cytotoxin associated gene A.

Discussion

H. pylori is well known for its genetic diversity and geographical differences, which may be associated with compound clinical disease outcomes (7). In the present study, the epidemiological characteristics of major virulence genes of H. pylori were investigated in Guizhou province. H. pylori was cultured from clinical samples, and virulence genotypes of cagA, vacA and iceA were examined by PCR assays. The results showed the prevalent strains of H. pylori in Guizhou province were cagA-positive, vacA s1c/m2-positive and iceA-positive. The results describe the prevalence of H. pylori strains in Guizhou province between January 2015 and December 2016, and provides an experimental basis for molecular epidemiological investigations of H. pylori in this region.

Epidemiological investigations have shown that the cagA genotype varies markedly worldwide. The prevalence of cagA among H. pylori in different regions varies between 50 and 60% in certain western countries to almost 100% in East Asia (19). In the present study, it was shown that cagA genotypes were detected in all strains in Guizhou province. In another study in this region, Zhou et al reported that 30 H. pylori strains isolated from gastric cancer were cagA-positive, with cagA having a positive rate of 100% (35), which was consistent with the results of the present study. The vacA genotypes also vary geographically. The vacA s1a genotype is common in South Asia, the s1b genotype is common in Latin America and Africa, and s1c is common in East Asia (24,36,37). The vacA m1 genotype is common in North Asian countries, including Japan and South Korea, whereas the m2 genotype is predominant in Southeast Asia, including Taiwan, China and Vietnam (20). The results of the present study showed that the predominant vacA genotype was s1c/m2 in Guizhou province. Notably, the iceA genotype also varies among different regions. A previous meta-analysis showed that the prevalence of iceA1 was significantly higher in East Asian countries than in western countries, whereas the prevalence of iceA2 was higher in western countries than in East Asian countries (26). Consistent with this finding, the iceA1 genotype was predominant in Guizhou province in the present study.

Although Guizhou province is a multi-ethnic society, there was no ethnic specificity of H. pylori infection. This differs from Malaysia, which has three major ethnic groups, namely, Malay, Chinese and Indian; vacA s1a/m2 has been detected in all of these ethnic groups, whereas vacA s1c/m2 was only isolated from Chinese patients (38). Therefore, vacA genotypes were associated with ethnic groups in Malaysia. In the present study, as no predictive value of these H. pylori virulence genes was identified in Guizhou province, there may be no need to classify patients according to ethnicity during treatment, with patients of different ethnicities offered the same diagnosis and treatment strategies.

H. pylori infection can result in compound gastroduodenal diseases, including gastritis, peptic ulcer and gastric carcinoma. The present study did not find an association between H. pylori virulence diversity and clinical disease outcomes in the Guizhou region. Zhou et al reported that H. pylori infection may induce the demethylation of lactate dehydrogenase, dihydrolipoamide dehydrogenase and calmodulin genes, and increase methylation of the Ran-specific GTPase-activating protein gene, which leads to dysfunctional gene expression in gastric cancer tissues and cells (35). A number of reports have shown that infection with a higher number of cagA EPIYA-C motif strains was associated with increased gastric inflammation and atrophy in western countries (39–41). However, the genetic diversity of H. pylori virulence genes is not associated with disease progression in certain eastern countries (9,16). Matsunari et al reported that, in the Bhutan region, 209 isolate strains, in which >50% of the strains had multiple EPIYA motifs repeats in East Asian-type cagA-ABD, are associated with atrophic gastritis and gastric cancer (42). An association between vacA genotypes and gastric precancerous or gastric carcinoma was observed in Brazil (39). However, no correlation has been found between vacA genotypes and diseases in Japan or Sweden (9,41). Additionally, an association has been reported between the iceA1 genotype and peptic ulcers, and the iceA2 genotype and gastritis (26). Among the 73 isolates in the present study, no association was found between the iceA1 or iceA2 genotypes and clinical disease outcomes.

Among the 73 patients, none were diagnosed with gastric cancer; only 14 gastric biopsy samples were obtained from patients with gastric cancer at the First Affiliated Hospital of Guizhou Medical University and the People's Hospital of Qiannan Autonomous Prefecture between January and December 2016, however, H. pylori isolation was negative. This may be associated with reduced Helicobacter abundance and overrepresentation of bacterial genera.

Current knowledge of bacteria other than H. pylori in the human stomach remains limited. The investigation of the gastric microbiota in healthy individuals or disease states is lacking due to limitations of culture conditions and the collection of gastric mucosal specimens. With the development of molecular biology and bacterial 16S rRNA gene identification technology, the constitution and diversity of gastric flora has been gradually identified. Notably, You et al reported a microarray used to compare the genomic profiles of strains isolated from patients with gastroduodenal diseases in the Heilongjiang province of China, and their findings may provide insight into novel biomarkers for the prediction of gastric diseases (43). Novel genetic variations may be examined in our future investigations using similar approaches. The gastric flora may be important roles in human health and disease, which remain to be elucidated. The composition of the gastric flora is dynamic and affected by several factors, including H. pylori infection and combination therapy with antibiotics and proton pump inhibitors. The gastric flora may also affect the development of gastric diseases following H. pylori colonization. H. pylori infection in Guizhou province may lead to various diseases without specificity. This may be explained by gastric flora in the stomach of local residents. Finally, the clarification of genotypes of gastric flora from different areas may facilitate clinical therapeutics.

Acknowledgements

The authors would like to thank Professor Jie Yang, Professor Yonghong Zhang, and Dr Chen Pan from the Department of Gastrointestinal medicine (The First Affiliated Hospital of Guizhou Medical University) for case selection.

Funding

This study was supported by the National Natural Science Foundation of China (grant no. 81460314).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

ZC conceived, designed and supervised the experiments. LY, FL, CG and QW performed the experiments. KP, LX, YX and YC collected the samples. ZC and LY performed the data analysis and wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

All protocols were approved by the Ethical Committee of the First Affiliated hospital of Guizhou Medical University. All procedures contributing to the study complied with the Declaration of Helsinki. Written informed consent was obtained from all patients.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

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References