Key words

Key words:
Helicobacter pylori, Biochemical Diagnosis, Antibiotic Susceptibility, Pan drug resistant, Cytotoxin-Associated , Vacuolating Cytotoxin , Genotypes.

1. INTRODUCTION

Helicobacter pylori is a fastidious gram-negative, spiral-shaped bacterium with lophotrichous flagella (Ashwini et al., 2014; Ndip et al ., 2008). The organisms colonize several areas of the stomach and duodenum and are specifically adapted to colonize and survive the hostile acidic gastric environment. More than half of the world’s population harbors this bacteria in the upper gastrointestinal tract (Parreira et al., 2013) and the infection could reach more than 70% in developing countries (De Martel et al ., 2012), more than 80% of persons infected with the bacterium are asymptomatic (Tanih et al., 2010). The Infection may result in a number of gastroduodenal disorders, including Gastric and duodenal ulcers. Also associated with gastric carcinoma and Mucosa Associated Lymphoid Tissue (MALT) lymphoma (de Bernard and Josenhans , 2014). Gastric carcinoma is considered as the third most common cause of death worldwide (Pinto-Ribeiro et al., 2016). Successful therapy of H. pylori infections consists on triple therapy, including aproton pump inhibitor or bismuth and any of the two antibiotics such as amoxicillin (AMX), clarithromycin (CLA), metronidazole (MNZ) and tetracycline (TET). However, H. pylori resistance to most of these commonly used antibiotics is raising worldwide( Iwanczak, and Iwanczak, 2012) . The genomic and phenotypic features of different strains permit the expression of virulence factors which allow some strains, rather than others, to cause disease (Meine et al., 2011) .H. pylori CagA is a high immunogenic protein associated with cell injury and more severe clinical outcomes, such as gastric adenocarcinoma and duodenal ulcer ,also affect the host cell in several aspects such as the formation of gastric epithelium cell pedestals, the change of the cytoskeleton, affect on cell proliferation and differentiation, and stimulating the gastric epithelium cells to secrete IL-8 (Boonyanugomol et al., 2011, Boonyanugomol et al., 2013). Several studies indicated that the CagA-positive H. pylori strains are directly associated with acute gastritis, gastric ulcer, and gastric cancer development (Matos et al., 2013, Azuma, 2004). VacA is the second most important studied H. pylori virulence factor. Furthermore to enhancing vacuolation, VacA cause several cellular activities, including membrane-channel forming, cytochrome c liberate from mitochondria leading to apoptosis (Akazawa et al., 2013), and attach to cell-membrane receptors followed by induction of a proinflammatory response (Amieva and El-Omar, 2008). VacA disrupts the balance of cell proliferation and death by affecting genes that regulate the cell cycle. In this study we aimed for molecular confirmation of H. pylori using vacA, cagA virulence genes.

2. MATERIAL AND METHODS

2.1. Collection of Gastric Biopsies

The biopsy specimens were collected during the period of April 2015 to April 2016 from 25 dyspeptic patients clinically expected to have H. Pylori infection. Two biopsies were taken from the antrum of the stomach of each patient by forceps of Olympus at Gastrointestinal Endoscopy Unit of Tanta University Hospital. Personal history from each patient were taken include: name, age, sex, residence. Patients taking antimicrobial drugs , proton pump inhibitor, and or bismuth salts two weeks prior before endoscopy were excluded.
One biopsy was placed directly in sterile tubes containing 1ml of phosphate buffer saline solution (PBS) or placed in 2ml Tryptical Soy Broth (TSB) as a transport medium and transported to the Medical Microbiology Laboratory and processed for culture as soon as possible. Second biopsy used for histopathology was transported with 10% buffered formalin for at least 24 h to the histopathology laboratory, Faculty of medicine, Tanta University.

2.2. Processing of the specimens for cultivation
Specimens were processd within less than one hour. Each biopsy of mucosal tissue was finely minced within their transport medium in a tissue grinder (mortar) with the aid of a sterile fine glass rod until the formation of homogenate and then processed for culture. Small fragments of the grinded biopsy were placed directly into Christensen’s urea medium for rapid urease test.

2.3. Culture of processed biopsy specimens

The processed biopsies were inoculated onto selective Columbia blood agar (Oxoid, England ) plates supplemented with 5% defibrinated sheep blood ,selective supplement(Dent) include, Trimethoprim (5 mg/L), Vancomycin (10 mg/L), Cefsulodin (5 mg/ L), and Amphotericin B (5 mg /L) .Plates were incubated at 37?C in an anaerobic jar for 3-10 days under microaerophilic conditions (85% (v/v) N2, 10% (v/v) CO2 and 5% (v/v) O2) by using Campygen kits (Oxoid , Basingstoke , UK), colonies of H. pylori formed on selective plates were confirmed by positivity for urease, catalase, and oxidase tests (Allahverdiyev et al., 2015). Additionally, Gram staining and microscopic analysis was performed to verify the presence of Gram-negative spiral rod bacteria.

2.4. Confirmation of the suspected H. pylori colonies
2.4.1. Colony morphology
The bacterial colonies of H. pylori were recognized by visual examination as small (1 mm or less in diameter) dome-shaped colonies or as a fine, translucent lawn (Farinha and Gascoyne , 2005) .
2.4.2. Gram staining
Gram stained smear from the obtained colonies that showed the presence of gram negative spiral shaped microorganism under light microscopic was considered as H. pylori (Cheesbrough, 2002).

2.4.3. Biochemical tests

The bacterial culture was biochemically identified using catalase, oxidase, and urease tests Al-Sulami et al , 2008 ; MacFaddin, 2000.

2.4.3.1. Rapid urease test

The urease test was applied according to MacFaddin (2000). The biopsy fragments were inoculated on the entire surface of Christensen urea agar slants or inoculated in Christensen urea broth. The tubes were inoculated at 37°C in the incubator. The formation of purple color was observed after 4 h.

2.4.3.2. Catalase test

The isolates were tested for catalase production by using slide method in which the sterile wire loop is
Stacked on the surface of the pure colony and transferred to a slide and one drop of 3% hydrogen peroxide
were put on the organism on the slide .The development of oxygen bubble indicate positive reaction (Abu-Sbeih et al., 2014).

2.4.3.3. Oxidase test
By using oxidase kits (Oxoid , UK) in which the colonies isolated on blood agar plate were examined by streaking on the oxidase kit. H. pylori colonies rapidly produce a purple color in few seconds.

2.4. 4. Histopathologic examination

This test was done by the laboratory technical in histopathology laboratory and read by the doctor following standard technique (Abu-Sbeih et al., 2014). The gastric biopsies were fixed in 10% buffered formalin for 24 h and then embedded in paraffin. The tissues were cut at 0.3 micron, de-paraffinized andhydrated in descending grades of alcohol, and stained with modified Giemsa stain to determine the presence of H. pylori and for evaluation of the pathological changes to the gastric mucosa. The H. pylori were identified as spiral shaped bacteria on the luminal surface of the gastric epithelial cells .
2.5. Antibiotic susceptibility test
In the present study antibiotic discs of different classes (Oxoid , England) ) were used to determine
the in vitro sensitivity of H. pylori clinical isolates to eleven antimicrobial agents commonly used in treatment of H. pylori. include: clarithromycin (15 ?g), tetracycline (10 ?g), ampicillin (10 ?g ) ,amoxicillin (10 ?g), amoxicillin / clavulanic acid (30?g), metronidazole (5?g) erythromycin ( 15 ?g), rifampin (5?g), ciprofloxacin (5 ?g), levofloxacin(5 ?g), and Gentamicin (10 ?g) were tested on nine clinical bacterial isolates using disk diffusion method . The bacterial suspension was prepared using few separate fresh colonies of each isolate which suspended in 1 ml phosphate buffer. Each suspension was diluted to achieve turbidity equivalent to that of no 3 McFarland turbidity standard (1x109cfu/ml) .Muller Hinton (MH) (Oxoid) agar plates containing H .pylori selective supplement were inoculated by swabbing the surface from each of previous suspensions using sterile cotton swabs (Kulsuntiwong et al., 2008) . Under aseptic condition, the antibiotic disks were applied to prepared plates using sterile forceps with 1cm part, and they were pressed gently in site to ensure good contact with the surface of the medium. The plates were incubated under microaerophillic conditions for 72 hours at 37ºC. After 2 days, zone of inhibition (mm) was measured and values were interpreted as sensitive(S), intermediate (I), resistant (R) for each antibiotic based on EUCAST and the Clinical and Laboratory Standards Institute (CLSI, 2013) .

2.6. Molecular Techniques
For the isolation of the DNA from the bacterial cells, transparent colonies (1-2 mm in diameter) were collected in 1.5 mL microcentrifuge tubes containing 1 mL PBS. The tubes were centrifuge at 5000 x g for 5 min, discard the supernatant. Genomic DNA was extracted from pellet by Quick- DNA ™ Mini prep kit ( Zymo Research ,USA) according to the manufacturer’s instructions. The isolated DNA was stored at -20 °C until tested. The genes amplification by thermal cycler (Hettich, Germany) and detection by agarose gel electrophoresis (Pharmacia, Sweden) were performed at Molecular Biology Unit, Faculty of Medicine, Al-Azhar University, Cairo.
2.6.1. CagA and VacA genes amplification by PCR:
PCR assay was performed in a total volume of 50 µL with specific primers (Table 1), the following cycling parameters were used; an initial denaturation step at 94oC for 4 minutes and 35 cycles of denaturation at 94oC for 1minute, annealing at 59oC for 1minute for cagA and 52°C for vacA, extension temperature at 72°C for 1minute and afinal extension step at 72°C for 10 minute (Bindayna et al., 2006). Each PCR product was separated on 2% agarose gel stained with ethidium bromide, and a 50 bp ladder was used as DNA molecular weight standard. In each PCR assay, a negative control (lacking DNA) was included.
Table 1: PCR primers for amplification of CagA and VacA genes (Falsafi et al., 2009)

Primers Primers sequence(5\-3\) Product size (bp)
CagA F: 5’AAT ACA CCA ACG CCT CCA-3′
R: 5’TTG TTG CCG CTT TTG CTC TC-3′ 400
VacA (s1/s2) F: 5’ATG GAA ATA CAA CAA ACA CAC-3′
R: 5’CTG CTT GAA TGC GCC AAA C-3′ 259/286
VacA (m1/m2 F: 5’CAA TCT GTC CAA TCA AGC GAG-3′
R: 5’GCG TCT AAA TAA TTC CAA GG-3′ 570/642