Prevalence of H. pylori Isolates and Genotypes and CagA Gene

1. Introduction

The best-known gastric Helicobacter species is Helicobacter pylori which is a slightly curved, microaerophilic Gram-negative. This microorganism has established a niche in the stomach of more than half of the world’s human population and become a dominant species of the human gastric microbiome. H. pylori colonization causes persistent inflammatory responses.H. pylori-induced gastritis is the strongest singular risk factor for cancers of the stomach however only a small proportion of infected individuals proceed to the development of malignancy. It is still unknown what factors are responsible for the different outcomes ranging from asymptomatic gastritis, peptic ulcer disease (PUD) to gastric cancer (GC). The changes in the epidemiology of these different demonstration of H. pylori infection suggest an environmental factor, an interaction between environmental factor and the host, or a change in prevalence of strains differing in virulence H. pylori has two phenotypic characteristics among strains are the vacuolating cytotoxin (vac) and cytotoxic associated gene A (vacA) protein which encoded by the and cagA genes respectively. cagA and genes are considered as candidates for the identification of strains with enhanced virulence. For instance, gastric mucosa from patients infected with cagA–positive H. pylori strains which containing a 40-kb cag pathogenicity island (which includes cagA gene) typically exhibits more severe inflammation than that of gastric mucosa infected with cagA-negative strains (Jafari et al., 2008). Despite the gene is present in most H. pylori, but the product may not be expressed in all cases. They have important attempt to described and classify distinction in the gene and to associate specific genotypes with different H. pylori-associated diseases The protein contains three distinct segments that exhibit variation within the amino-terminus. These areas of variation are broadly defined as the signal (s), intermediate (i), and middle (m) regions. Two primary variants have been described for signal region (s1 or s2) and middle region (m1 or m2) while three variants has been reported for intermediate region (i1, i2, or i3) (Jones et al., 2010). The m region affects host cell tropism of toxins. Encoding the m1 region are toxic to a broader range of host cells compared to that of m2 region. The i region which determine the vacuolating capabilities is positioned between the s and m region and is the most recent region to be described. The i1 variants of have been shown to have stronger vacuolating activity than i2 variants (Jones et al., 2011). Atherton et al. (1995) reported three of the four possible subtypes with the exeption of s2-m1 variant. The presence of the s2-m1 variant was later reported but with very rare prevalence (0 to 3%). Despite variants have been described, but is still remains unclear whether the genotypes are useful markers for the prediction of clinical outcomes. Initial reports indicated that the s1 and i2 genotype would be found in close association with clinical outcomes in Western countries (Jafari et al., 2008); however the prevalence of this genotype was extremely high (almost 100%) in East Asian countries irrespective of the clinical outcomes (Jafari et al., 2008). The prevalence of H. pylori infection is about 50% of the world’s population and has been reported to be 40 to 60% in Malaysian (Goh et al., 2009).

The purpose of this study is to identify the prevalence of H. pylori isolates in patients with a full spectrum of clinical demonstration of H. pylori infection to evaluate whether the assumed associations with genotypes and cagA gene would be applicable to patients with H. pylori infection in Malaysian populations.

2. Material and methods

Isolation and culture of H. pylori

Gastric biopsies were obtained from each patient by using endoscopy. Each specimen was placed in a transport medium and sent to the laboratory immediately. The specimens were then incubated onto chocolate (selective/non-selective) agar containing 10% sheep blood for 3 days at 37?C under microaerobic conditions (10% CO2, 5% O2, 85% N2). All H. pylori strains were positive for urease, oxidase, catalase and were identified by Gram staining under light microscopy. Colonies directly harvested from the plates were used for DNA extraction. Endoscopic findings were also recorded and based on the information patients were divided into two groups. Group I patients had duodenal or gastric ulcer (Peptic Ulcer Disease, PUD) and group II patients had Non-Ulcer Dyspepsia (NUD).

Population studied

A total of 209 gastric biopsies were collected from patients presenting with symptoms of gastroduodenal diseases during July 2011 to August 2012 at the University of Malaya Medical Centre (UMMC). Four biopsy specimens were taken from the greater curve of the antrum, body, cardia and Esophagous, one was used for histological examination and one for H. pylori culture.

Genomic DNA extraction

Bacteria from both the 10 single colonies and the sweeps were resuspended in 1.2 ml Elution buffer (EB). Bacterial pellets were obtained by centrifugation at 5,233 ?gfor 5 min, and genomic DNA was extracted by using a Qiagen kit. The amount of DNA was calculated by spectrophotometry.

Detection of H. pylori and genotyping of 16sRNA, cag A, E, L and vac genes and EPIYA motifs by PCR

In this study PCR was used to detect the H. pylori specific 16sRNA gene for confirmation of H. pylori isolates, the virulence-associated vacA mosaic structure and the presence of cag A, E, L genes. All primer sets were selected from the published literatures (Table 1). The PCR was carried out in a volume of 25 µl including100 ng of genomic DNA, 20 pmol of each of primers (Table 1), 5 µl of i-PCR 5X Master Mix (iDNA, Singapore) which contain 0.1 unit/µl 5 µl of i-PCR 5X Master Mix (iDNA, Singapore) which contain 0.1 unit/µl of Taq DNA Polymerase, 1 mM of dNTP’s, 10% of glycerol and 7.5 mM of MgCl2. Negative control tubes included all materials except template DNA. The sequences of the primers are listed in Table 1. PCR products were checked on 2.0% agarose gels with 100 bp ladder size markers. Polymerase chain reaction (PCR) for the EPIYA motifs was performed in separate reactions according to the

method described by Argent et al. (Mitchell et al., 2009). using the common forward primer cag2 with one of three reverse primers, cagAP1C, cagAP2TA, and cagAP3E, for the amplii¬?cation of EPIYA-A, -B, and -C and -D, respectively.

Table 1. Oligonucleotide primers used for cagA/E and L, vacA alleles and EPIYA motifs

Primer

Sequence

PCR conditions

PCR product size (b.p)

Ref

S1/S2-F

5’-ATG GAA ATA CAA CAA ACA CAC-3’

95^oC, 5 min; 35x (95^oC, 20 s; 52^oC, 20s; 72^oC, 40s); 72^oC, 7 min

259/ 286

3

S1/S2-R

5’-CTG CTT GAA TGC GCC AAA C-3’

M1/M2-F

5’-CAA TCT GTC CAA TCA AGC GAG-3’

95^oC, 5 min; 35x (95^oC, 20 s; 52^oC, 20s; 72^oC, 40s); 72^oC, 7 min

259/ 286

M1/M2-R

5’-GCG TCT AAA TAA TTC CAA GG-3’

I-F

5’-GTT GGG ATT GGG GGA ATG CCG-3’

–

–

I1-R

5’-TTA ATT TAA CGC TGT TTG AAG-3’

95^oC, 5 min; 35x (95^oC, 20 s; 55^oC, 20s; 72^oC, 40s); 72^oC, 7 min

426

I2-R

5’-GAT CAA CGC TCT GAT TTG A-3’

95^oC, 5 min; 35x (95^oC, 20 s; 55^oC, 20s; 72^oC, 40s); 72^oC, 7 min

432

CagA-F

5’-GAT AAC AGG CAA GCT TTT GAG-3’

95^oC, 5 min; 35x (95^oC, 30 s; 50^oC, 30s; 72^oC, 1 min); 72^oC, 7 min

349

CagA-R

5’-CTG CAA AAG ATT GTT TGG CAG-3’

CagE-F

5’-TTG AAA ACT TCA AGG ATA GGA TAG AGC-3’

95^oC, 5 min; 35x (95^oC, 30 s; 53^oC, 30s; 72^oC, 1 min); 72^oC, 7 min

508

CagE-R

5’-GCC TAG CGT AAT ATC ACC ATT ACC C-3’

CagL-F

5’-AAG CGT CTG TGA AGC AGT GA-3’

95^oC, 5 min; 35x (95^oC, 30 s; 55^oC, 30s; 72^oC, 1 min); 72^oC, 7 min

433

CagL-R

5’-GAC CAA CCA ACA AGT GCT CA

26KDA-F

5’-TGG CGT GTC TAT TGA CAG CGA GC-3’

98°C, 10 min;37x ( 92°C, 30s; 68°C, 1 min; 6x (92°C, 30s; 68°C, 1 min; 72°C, 2 min)

303

2

26KDA-R

5’-CCT GCT GGG CAT ACT TCA CCA G-3’

16S-F

5’-CTG GAG AGA CTA AGC CCT CC-3’

30x (95^oC, 30 s; 62^oC, 30s; 72^oC, 30 s)

109

16S-R

5’-ATT ACT GAC GCT GAT TGT GC-3’

s1a-F

5’-GTC AGC ATC ACA CCG CAA C-3’

95^oC, 5 min; 35x (95^oC, 30 s; 60^oC, 30s; 72^oC, 1 min); 72^oC, 7 min

190

s1a-R

5’-CTG CTT GAA TGC GCC AAA C-3’

s1b-F

5’-AGC GCC ATA CCG CAA GAG-3’

187

s1b-R

5’-CTG CTT GAA TGC GCC AAA C-3’

Cag2

5?-GGA ACC CTA GTC GGT AAT G-3?

95^oC, 5 min; 35x (95^oC, 30 s; 55^oC, 30s; 72^oC, 1 min); 72^oC, 7 min

550-800

EPIYA-A

5?-GTC CTG CTT TCT TTT TAT TAA CTT KAG C-3?

264

EPIYA-B

TTT AGC AAC TTG AGC GTA AAT GGG

306

EPIYA-C

5?-TTT CAA AGG GAA AGG TCC GCC-‘3

95^oC, 5 min; 35x (95^oC, 30 s; 55^oC, 30s; 72^oC, 1 min); 72^oC, 7 min

501

EPIYA-D

5?-AGA GGG AAG CCT GCT TGA TT-‘3

495