Molecular characterization and antibiotic resistance profiles of H Molecular characterization and antibiotic resistance profiles of H. pylori clinical strains in Jordan Dr. Luay Abu-Qatouseh PhD Medical and Molecular Microbiology and Immunology Microbiology section Head-Jordan Company for Antibody Production

History Isolation of H. pylori from peptic ulcer patients in 1982 In 2005, Nobel Prize Barry J. Marshall J. Robin Warren

Pathology and diseases Majority of infected patients do not develop clinically significant diseases Significant manifestations peptic ulcer disease (PUD) gastric and duodenal ulcers chronic gastritis mucosa associated lymphoid tissue (MALT) gastric adenocarcinoma

Epidemiology Estimated 50-60% of the world population is infected Person to Person Transmission fecal-oral, oral-oral, gastro-oral Increased risk of infection younger age underdeveloped countries lower socioeconomic status

Epidemiology Developed Countries: Developing Countries: Lower rates of infection and increased gradually with age ~50% by 50-60 yrs ~1/3 eventually have peptic ulcer disease(PUD) 70% gastric ulcer cases colonized with H. pylori Low socioeconomic status predicts H. pylori infection Developing Countries: Higher rates ( particularly in childhood, 50-60% by age of 10 yrs and ~90% in adults) Most adults infected but remain asymptomatic 70-90% of patients with duodenal ulcer have H. pylori infection

Virulence and adaptive mechanisms Gastric inflammatory potential LPS Urease IL-8 Altered gastrin-gastric acid homeostasis Disruption of mucosal barriers Cytotoxin associated genes products Vaculating cytotoxins Phospholipases Mucinase Penetration and colonization Motility Bacterial adhesins and OMPs Adaptive enzymes (catalase, SOD, Heat shock proteins Immune evasion Suppression of immune response Resistance to killing by PMNs Immune mimicry (Lewis antigens)

When to test for H. pylori Strongly Recommended Dyspepsia peptic ulcer disease Gastric MALT lymphoma Following gastric cancer resection or peptic ulcer surgery First-degree relative with gastric cancer Long-term Non-steroidal anti-inflammatory drugs (NSAID) therapy Advisable Family history of duodenal ulcer Family members with H. pylori infection

Diagnosis Invasive (Endoscopy and biopsies) Histopathology (Sydney Grading) Rapid urease test Culture PCR Non-invasive Serology Stool Antigens and PCR carbon-labeled urea breath test (13C-UBT)

Advantages Disadvantages Test Advantages Disadvantages Endoscopy Inspection of ulcers and neoplasms Invasive, expensive, not conclusive Histology Allows direct Visualization and grading Sens 95%, spec 95-98% Useful for eradication Time consuming Requires high bacterial load Stomach part dependent Culture Most specific (100%) Allows antimicrobial susceptibility testing Lower sensitivity (75-90%) Difficult Urease Rapid Sens 95%, spec 90-95% False positive is samples other than biopsies Not useful for eradication follow up Serology Non invasive, low cost Sens 85%, spec 80% Detects previous infection No correlation to pathology or antimicrobial resistance Urea breath test Non invasive , rapid Sens 95%, spec 95% Useful for assessing eradication Expensive Stool Antigen Sens 85-90%, spec 95% Not Very sensitive PCR Allows detection of Viable and non-Viable cells and antibiotic resistance Sens 90%, spec 98% No correlation to pathology and lesion costly

Treatment Standard First‐line therapy Alternative First‐line therapy Triple therapy : PPI, amoxicillin, clarithromycin or PPI, amoxicillin, metronidazole (In infection resistant to clarithromycin) or PPI, clarithromycin, metronidazole (In patients allergic to penicillin) Alternative First‐line therapy Triple therapy : PPI, levofloxacin, amoxicillin (In dual resistance to clarithromycin and metronidazole) Quadruple therapy : PPI, bismuth, metronidazole, tetracycline (In areas where the resistance to clarithromycin or metronidazole is >20% or in patients with recent or repeated exposure to clarithromycin or metronidazole)

The problem $6 billion / yr in health care costs due to peptic ulcer disease Increase resistance to antibiotics led to increase rate of triple therapy failure Misuse of antibiotics exaggerated the problem and increased the rate of infection Increase rate of re-infection Difficulty in performing antimicrobial susceptibility testing and improper or Variation in interpreting the test results in relation to resistance criteria

The aim of the study Determination of antibiotic resistant rates of the clinical isolates Molecular characterization of antibiotic resistance genes of the clinical isolates and their association with virulence

The study Samples and patients: (JUH) Gastric biopsies collected from patients with symptoms of gastro-dudenal diseases referred to JUH during May-Dec 2012 Testing (MONOJO Microbiology Lab and faculty of Pharmacy-JU) Isolation and Identification of H. pylori Urease: Commercial CLO test Culture:7% laked horse blood with dent supplement Biochemical assays: Catalase, oxidase and urease PCR: 16S rDNA Antimicrobial susceptibility testing and MIC determination (standard disc diffusion assay, E-test, and Agar dilution assays according to the CLSI standards) Molecular typing (CagA, VacA, ….)

Results Prevalence of H. pylori in the clinical samples 83 samples 32 (39 %) urease (+) 13 (40%) culture (+) 19 culture (-) 51 urease (-) 51 culture (-)

Left: Colonial morphology of H Left: Colonial morphology of H. pylori on 7% laked horse blood Agar; Small translucent colonies Right: Gram Staining of H. pylori using Carbulfuschin as counter stain; Gram negative curved rods.

Confirmation of H. pylori by standard 16S rDNA of H. pylori PCR NC S1 S2 S3 S4 PC 1000bp 521bp 500bp M: 100 bp DNA ladder, NC: negative control, S1-S4: clinical isolates PC: H.pylori control strain (NCTC 11916)

Susceptibility to standard antibiotics Strain Number Standard Antibiotics AML CIP LEV CLR MTZ Cl. St. 1 S (<0.015) S (0.015) S (0.008) S (0.5) Cl. St. 2 S (0.12) R(16) Cl. St. 3 S (0.03) R(32) Cl. St. 4 R (4) S (0.25) R(64) Cl. St. 5 S (0.06) Cl. St. 6 Cl. St. 7 Cl. St. 8 R (16) R (32) Cl. St. 9 S (8) Cl. St. 10 Cl. St. 11 Cl. St. 12 Reference strain

MIC determination of the clinical strains against standard antibiotics using both E test method and the standard agar dilution assay

Resistance to antibiotics

Genotyping of virulence genes (CagA and VacA) M NC PC S1 S2 S3 S4 S5 Positive cagA (46%) Positive VacA m1 (23%) Positive VacA m2 (55%) Negative VacA (22%) Dual VacA m1,m2 (8%) CagA+/VacA m1 (23%) CagA+/VacA m2 (23%) 16S rDNA VacA m2 VacA m1 CagA M: 100 bp DNA ladder, NC: negative control, PC: H. pylori control strain (NCTC 11916), S1-S4: clinical isolates. 16s rDNA= 521bp, VacA m2=325 bp, VacA m1=290 bp, CagA= 183 bp

Molecular mechanisms of metronidazole resistance rdxA gene: Oxygen insensitive NADPH nitroreductase MTZ resistance due to deletion in rdxA gene was reported in 15% 850 bp (normal gene) 650 bp (gene deletion)

Strain   Genotype NCTC CagA+, VacA m1, Rdx 600 S1 CagA-, VacA m2, Rdx 800 S2 CagA+, VacA m2, Rdx 800 S3 CagA+, VacA m1,m2, Rdx 800 S4 S5 S6 S7 CagA+, VacA m1, Rdx 800 S8 CagA-, VacAm2, Rdx 800 S9 S10 S11 CagA+,VacA m1,Rdx 600 S12 CagA-,VacA m2,Rdx 800

Conclusions Infection rate of H. pylori among Jordanian patients with gastro-dudenal diseases is high Metronidazole resistance is dramatically high Increased resistance toward ciprofloxacin and clarithromycin is reported Concern should be taken when using first line antibiotics in the treatment of infection caused by H. pylori The eradication of H. pylori by the commonly used antibiotics must be studied

Conclusions Variation of the genetic makeup of the virulence of the strains is observed No correlation between virulence genotypes and antibiotic resistance Mechanisms of MTZ resistance other than mutations in rdxA gene should be investigated

Publications and Patents Abu-Qatouseh, L.F., Boutennone, H., Boussouf, L., Madani, K., Shihab, P., Al-Qaoud, K., 2014. In Vitro anti-Helicobacter pylori and urease inhibitory effects of polyphenolic extracts of local herbs from Algeria. The International Arabic Journal of Antimicrobial Agents 3. Al-Qaoud, K.M., Shihab, P.A., Abu-Qatouseh, L.F., Lowe, C.R. Immunized Camel Milk-Based Composition for the Treatment or Prevention of Gastrointestinal Infections. 2012. Google Patents. Ref Type: Generic Molecular characterization and antibiotic resistance profiles of H. pylori clinical strains in Jordan (draft).