1. 서울대학교 대학원 의과학과
분당 서울대학교 병원
Influence of Efflux Pump Inhibitors (EPIs) on
Clarithromycin- and Moxifloxacin-Resistant
Helicobacter pylori
학생연구원 이연석 (Austin Lee)

2. Contents
Aim
Background
Method
Results
Discussion

3. Aim
 To investigate the contribution of efflux system in CLA- and MOXI-
resistant H. pylori strains by testing the efficacy of EPI
 To compare the EPI efficacy between CLA- and MOXI-resistant
strains
 To investigate specific mutations within these resistant strains and
find possible relationships between the mutation and resistance

4. Helicobacter pylori
 Gram-negative, microaerophilic bacterium
 Causes various gastroenterological diseases
 Antibiotics as treatment  resistance 
3/16

5. Moxifloxacin
 Fluoroquinolone  interrupts DNA replication; dual targets:
- Topoisomerase II (DNA gyrase)
- Topoisomerase IV
 Resistance  mutation in:
- QRDR (Quinolone Resistance-Determining Region) of gyrA
- Asn-87 or Asp-91 point mutations in gyrA
 N.B. gyrB mutations rarely occur and have little impact on
fluoroquinolone resistance (Lee, J.W. et al., 2011)

6. Clarithromycin
 Macrolide antibiotic  inhibits translation of peptides; target:
- Subunit 50S of bacterial ribosome
 Resistance  point mutations in 23S rRNA
- T2182C, A2143G or A2223G (also A2144G, T2190C, C2195T)
 ALSO: RND (Resistance-Nodulation-cell Division)
efflux pump mechanism
 4 RND families have been identified
- HP0605-HP0607 (Hef ABC)
- HP0971-HP0969 (Hef DEF)
- HP1327-HP1329 (Hef GHI)
- HP1489-HP1487 (-)
5/16

7.  E.g. Phenyl-Arginine-Beta-Naphthylamide (PAβN)
 Effective against AcrAB-TolC system in E. coli and
MexAB-Oprm system in P. aeruginosa
6/16
Efflux Pump Inhibitor (EPI)

8. Methods: MIC Test
 Agar dilution method
- MOXI & CLA: 0.25 – 32 μg/ml
- Spotted each strain on the plates
 Microaerophilic incubator (37℃; 5% O2; 10% CO2; 85% N2) for ~3 days
 Resistant if growth appeared on 1 μg/ml or more
O
4
1
2
3
6
5
4
7
MOXI/CLA 1
No Growth
Growth
MOXI/CLA 2 MOXI/CLA 4

9. EPI MIC Test
 Agar dilution method
- EPI: 10-60 mg/L
- Spotted MOXI/CLA-resistant strains on the plates
 Microaerophilic incubator for ~3 days
O
4
1
2
3
6
5
4
7
MOXI/CLA 1 +
EPI10
MOXI/CLA 1 +
EPI20
MOXI/CLA 1 +
EPI40
MOXI/CLA 1 +
EPI60

10. Mutation Check via PCR
 gyrA primers:
 23s rRNA primers:
 gyrA and 23s rRNA PCR cycle: 35 cycles of 1-minute denaturation at
94°C, 1-minute annealing at 57°C, and 1-minute extension at 72°C
 Agarose gel electrophoresis
 Purification of PCR-amplified products
 DNA sequencing (via MacroGen)
 Searched for any mutations
gyrA-Forward 5’-TTT AGC TTA TTC AAT GAG CGT-3’
gyrA-Reverse 5’-GCA GAC GGC TTG GTA GAA TA-3’
23s rRNA-Forward 5-TCA ACC AGA GAT TCA GT-3′
23s rRNA-Reverse 5’-TCC ATA AGA GCC AAA GC-3’
Wild Type
Asn-87
Asp-91

11. DNA Sequencing (CLA)
A2143G
A2223G
Wild Type

12. Results: MIC Test & Mutation
 Total moxifloxacin-resistant strains = 43
 Mutants = 36/43; non-mutant = 7/43
 Mutation  Asp-91: 22/36; Asn-87: 14/36
mutant
84%
non-
mutant
16%
Mutant vs Non-mutant
Asn-87
39%
Asp-91
61%
Asp-91 vs Asn-87

13. Moxifloxacin + EPI
0
5
10
15
20
25
30
35
MOXI MOXI + EPI
MICofMoxifloxacin(μg/ml)
MOXI vs. MOXI + EPI

14. Asn-87 vs Asp-91
14%
86%
Asn-87
MIC Reduction No MIC Reduction
73%
27%
Asp-91
MIC Reduction No MIC Reduction

15. CLA Results
 Total clarithromycin-resistant strains = 36 (all mutants)
 T2182C-only mutants= 4/36; Dual mutants = 32/36
- A2143G: 30/32; A2223G: 2/32
Dual
mutation
89%
Single
mutation
11%
Single vs Dual Mutation
A2143G
94%
A2223G
6%
A2143G vs A2223G

16. Clarithromycin + EPI
0
5
10
15
20
25
30
35
CLA CLA + EPI
MICofMoxifloxacin(μg/ml)
CLA vs. CLA + EPI

17. CLA MIC Reduction
75%
25%
Overall MIC Reduction
MIC Reduction No MIC Reduction
 All four T2182C-only mutants s
howed MIC reduction
 Both A2223G/T2182C dual mut
ants showed MIC reduction
 However, due to the limited nu
mber of stains, no solid conclus
ion can be made regarding the
influence of mutations on EPI e
fficacy

18. Summary
 21/43 strains showed MIC reduction
(49%)
- 2/14 Asn-87 mutants (14%)
- 16/22 Asp-91 mutants (73%)
- 3/7 non-mutants (43%)
 27/36 strains showed MIC reduction
(75%)
- 4/4 T2182C-only mutants (100%)
- 2/2 T2182C/A2223G mutants (100%)
- 21/30 T2182C/A2143G mutants (70%)
Moxifloxacin Clarithromycin
17/16
 MIC reduction after EPI addition in both antibiotics was confirmed

19. Discussion
 The efflux pump of H. pylori is associated with MOXI/CLA resistance in addition
to gyrA/23S rRNA point mutations.
 an attractive target for reversing drug resistance
 However, moxifloxacin exhibited contrasting efficacy of EPI for its two different
gyrA point mutations (i.e. Asn-87 and Asp-91)  further research required
 Even non-mutants (which were suspected of efflux pump overexpression) did
not have significant MIC reduction rate (43%)  there could be another strong
factor contributing to moxifloxacin resistance
18/16

20. What to do now…?
 Further investigate the difference between Asn-87 and Asp-91 point
mutations and their effects on EPIs
 Continue to look for T2182C and A2223G mutants to investigate their
relationships with efflux pumps
 Investigate the influence of different efflux pump gene clusters
(e.g. Hef ABC/DEF/GHI) on MOXI/CLA resistance
19/16

21. References
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clarithromycin resistance in Helicobacter pylori. Journal of Gastroenterology and Hepatology, 75-79. DOI: 10.1111/j.1440-
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22. Thank you for listening