1. 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

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

3. 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

4. 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

5. Epidemiology Developed Countries: Developing Countries:
Lower rates of infection and increased gradually with age
~50% by 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

6. 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)

7. 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

8. 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)

9. 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

10. 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)

11. 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

12. 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

13. 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, ….)

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

15. 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.

16. Confirmation of H. pylori by standard 16S rDNA of H. pylori PCRNC 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)

17. 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

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

19. Resistance to antibiotics

20. Genotyping of virulence genes (CagA and VacA)M NC PC S1 S2 S3 S4 S5
Positive cagA (46%)
Positive VacA m (23%)
Positive VacA m (55%)
Negative VacA (22%)
Dual VacA m1,m2 (8%)
CagA+/VacA m (23%)
CagA+/VacA m (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

21. 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)

22. 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

23. 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

24. 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

25. Publications and Patents
Abu-Qatouseh, L.F., Boutennone, H., Boussouf, L., Madani, K., Shihab, P., Al-Qaoud, K., 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 Google Patents. Ref Type: Generic
Molecular characterization and antibiotic resistance profiles of H. pylori clinical strains in Jordan (draft).