3.1. Data collection of the patients included within the study This study was carried out on 25 patients were complaining of upper gastrointestinal symptoms and clinically suspected to have H. pylori infection.
They were17 males and 8 females, and their ages ranged between 30 and 70 year (Table 2). In this study, 9 out of 25 cases were H. pylori positive with different endoscopic findings (Table3), the isolates were designated as HP1 to HP9. Table (2): Demographic features of the patients included within the studyDemographic features Patients` study No.
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= 25Age Range 30-70 Mean±SD Sex Male 17 (68.0%) Female 8 (32.0%)Residence Rural 20(80.0%) Urban 5(20.0%)Table (3): Frequency of H. pylori among cases with different endoscopic findingsNumber of H.
pylori positive cases Number of cases Endoscopic findings5 (45.4 %) 11 Duodenal ulcer2 (25.0%) 8 Gastric ulcer1 (20.
0%) 5 Gastritis and duodenitis1(100.0 %) 1 Gastric cancer9 (36.0%) 25 Total 3.2. Microscopic examination and biochemical tests Nine positive H.
pylori clinical isolates which cultured on the selective medium supplemented with selective antibiotic and 5% sheep blood, under microaeriphilic conditions. These isolates were identified according to colony morphology that appeared transparent, small colonies on surface of Colombia blood agar Fig (1). Microscopic examination of a bacterial film of H. pylori stained by Gram stain showed that H. pylori are Gram negative, curved rods and spiral in shape Fig (2). Biochemical tests showed that all of the 9 isolates were oxidase positive, catalase and urease positive Fig (3).
Fig (1). Appearance of colonies of Helicobacter pylori on Colombia blood agar that shown small, pinpoint, translucent to grayish white colonies after incubation at 37°C for 7 days and under microaerophilic conditions. Fig ( 2). Gram stained film of H. pylori under light microscope showing typical morphologic appearance Fig (3 ) . (A) positive catalase. (B) positive oxidase.
(C) Positive rapid urease test of an obtained gastric biopsy on Christensen’s urea medium : (1) after less than one hour, (2)after less than 5 hours. 3.3.
Histopathologic examination To confirm the infection with H. pylori histopathology was used The result indicated that 9 (36 % ) were histology positive and (64 %) were negative .Histopathology examination by Giemsa stain of positive H. pylori gastric biopsies showed the pathological changes to the gastric mucosa and indicates the presence of spiral shaped H. pylori colonization at the lumen of the gastric tissue Fig (4) and histopathology examination of negative H.
pylori gastric biopsies showed normal human gastric glands and absence of H. pylori bacteria from lumen of the gastric tissue fig (5). Fig (4) .Positive histopathological result .Part A.
Showed inflammatory cells by Giemsa stain (x 400) .The vertical arrow showed ulceration of gastric mucosa (discontinuous of epithelial cells).The horizontal arrow showed atrophic gastric glands and mononuclear cellular infiltration mostly by lymphocytes cells. Part B. Positive histopathological result by Giemsa stain (x 1000). The vertical arrow showed the presence of spiral shaped H.
pylori colonization at the lumen of the gastric glands. Fig (5) . Negative histopathological result by Giemsa stain (x 400) the lumen is clear (Normal human gastric glands) there is no bacteria and no inflammatory response. 3.4. Antibiotics susceptibility test of H.
pylori clinical isolates The isolates were tested for their susceptibility to eleven antibiotic disks of different classes by using the disk diffusion method. The antibiotic resistance profiles of the clinical isolates against eleven antimicrobial agents are presented in (Table 4). The result indicate that, the all 9 (100 %) tested bacterial isolates were resistant to amoxicillin, metronidazole, ampicillin, rifampin, gentamicin, amoxicillin / clavulanic acid and erythromycin .
On the other hand 6 (66.7 %) isolates were sensitive to levofloxacin and tetracycline (showed the lowest resistance) and remaining three isolates (33.3 %) were resistant.
While 4 (44.4 %) isolates were sensitive to ciprofloxacin and two isolates (22.2%) showed intermediate susceptibility and the remaining three isolates were resistant.
Only one isolate showed intermediate susceptibility to clarithromycin and other remaining eight isolates were resistant. The data showed in the (Table 4) revealed that among all clinical bacterial isolates, isolates No.3, No.5. No.8 were proved that to be pan drug resistant H.
pylori isolates recording 100 % resistance.HP9 HP8 HP7 HP6 HP5 HP4 HP3 HP2 HP1 IsolatesAntibioticsR R R R R R R R R AmoxicillinR R R R R R R R R Amoxicillin / Clavulanic acidR R R R R R R R R AmpicillinR R R R R R R R R RifampinR R R R R R R R R GentamicinS R S S R S R S S TetracyclineS R S S R S R S S LevofloxacinR R R R R I R R R ClarithromycinR R R R R R R R R MetronidazoleR R R R R R R R R ErythromycinI R S I R S R S S CiprofloxacinTable (4).Antibiotics susceptibility of H. pylori clinical isolates R: resistant; S: sensitive; I: intermediate.
3.5. Cytotoxin genes CagA and VacA genotypes in pan drug resistance Helicobacter pylori (HP3, HP5, HP8) isolates detected by PCR. PCR amplification of CagA and VacA genotypes was performed using specific primers for each toxin gene. Each PCR product was separated on 2% agarose gel and visualized under UV after staining with ethidium bromide , a 50 bp ladder was used as DNA molecular weight marker., a negative control (without DNA) was involved .The gel was examined by UV transilluminator, size and intensity of fluorescence bands of the DNA was compared with that of the ladder, the results showed the prescence of CagA gene in all three pan drug resistance H .
pylori isolates, while VacA m1 gene was detected in HP3, VacA s1 gene was detected in HP3 and HP8 , VacA m 2 gene was detected in HP5 and HP8 and VacA s2 gene was detected in HP5 Fig (6). Agarose gel electrophoresis for the amplicons of the CagA gene and different VacA gene subtypes: Lane 1 contains the DNA ladder (50 bp) ,Lanes 2, 5 and 8 contain the 400 bp CagA gene. While, lane 3 contain the 570 bp VacA m1 gene , Lanes 4 and 10 contain the 259 bp VacA s1 gene ,Lanes 6 and 9 contain the 642 bp VacA m2 gene ,lane 7 contain the 286 bp VacA s2 gene and lane 11 contains negative control.4. DISCUSSIONH. pylori is the most common human bacterial pathogen in the world .Gram negative spiral shaped bacteria that growing in microaerophilic conditions specially colonizes the gastric mucosa (Fiorentino et al.
, 2013).The current study was done on 25 patients complaining of different dyspeptic symptoms subjected to upper endoscopy. The percentage of infected people increases with age, in this study the highest percentage of patients was among people ranging from 40-60years, these agree with study made by (Abu-Sbeih et al., 2014). Gastric biopsies were collected from the antral region of the stomach of each patient, antral biopsy has been reported to be more sensitive in the detection of H. pylori when compared to that of the corpus ( Dursun et al.
, 2004 ). In this study a highest range of infection was found among males, out of 25 patients, 17males ,a study in contrary made by (Diab et al. , 2018) among the studied 60 H.
pylori infected patients, 40 patients were males and in the other study by (Majlesi et al., 2013) from153 patients, 89 males .From our results, it was showed that 9 (36 % %) from 25 cases were positive for H. pylori culture, a study made by (Jabbar and AL-Obaidi , 2015),among 45 biopsy samples only 15 (33.33%) were positive for H. pylori culture. Primary isolation of H.
pylori is a difficult process for routine laboratories , many laboratories have found the primary isolation of H. pylori from gastric biopsies is still problematic (Chomvarin et al., 2006). In this study, duodenal ulcer cases represented the major source of positive H. pylori isolates (45.4 %) and the remaining H.
pylori was found among gastric ulcer, gastritis and duodenitis and gastric cancer cases, these result disagree with (Al-Sulami et al., 2008) who isolated H. pylori from 100 % patients suffering from duodenal and gastric ulcer. From our result,the isolated H. pylori appeared on petri dish as small, round and transparent colonies, positive for urease, catalase and oxidase tests ,and which is supported by previous studies (Hassina and Ahmed , 2013 ; Jabbar and AL-Obaidi , 2015). The pathological profile of the gastric mucosa displayed presence of H.
pylori colonizedat the lumen of the gastric glands with chronic inflammatory infiltrate in lamina propria and atrophic gastric glands which is supported by previous studies (Trindade et al., 2017; Khalid et al., 2015; Garg et al., 2012).In the current study, almost all isolated H.
pylori isolates showed resistant to several used antibiotics, these results agree with (Aboderin et al., 2007) who reported that all the H. pylori isolates showed multiple acquired resistance as they were all resistant to amoxicillin, clarithromycin, metronidazole and erythromycin .From our results, most of H.
pylori isolates (66.7 %) were sensitive only to tetracycline , levofloxacin, ciprofloxacin (showed the highest susceptibility among all tested antimicrobial drugs ) this results is agree with a study by (Eng et al., 2015) reported that 70 % were susceptible to ciprofloxacin and levofloxacin, a study by (Eisig et al.
, 2011) who reported that 77% of H. pylori isolates were susceptible to levofloxacin but 100% were susceptible to tetracycline. In present study, DNA extracted from the pan drug resistant H . pylori isolates was used directly for detecting H. pylori vacA and cagA gens, we found that these pan drug resistant isolates are positive for vacA and cagA genes. A study by (Bachir et al., 2018) reported that no direct role of vacA or cagA genes in antibiotic resistance except for the metronidazole, which had relation with the presence of cagA genotype , Another report revealed that cagE and vacA S1 correlated with clarithromycin and metronidazole resisttance (Karabiber et al.
, 2014 ) . 5. CONCLUSIONSWe found elevated rates of primary antibiotic resistance among H. pylori clinical isolates of dyspeptic Egyptian patients. Further studies are required to con?rm this ?nding for optimization the clinical therapy of H. pylori infection.
We reported that vacA and cagA genotypes are present in H . pylori clinical isolates that showed pan drug resistance. In Egypt where H.
pylori is prevalent, more studies are needed to evaluate the association between virulence factors and antibiotic resistance that may give prediction of the improvement the eradication rates. Additional studies are required to evaluate the virulence factors of H. pylori with focusing on the role of CagA and VacA in H. pylori pathogenicity both in vivo and in vitro.
