Resistance Modifying Activity of Pomegranate Fruit Constituents against β-Lactamase Producing Gram-negative Clinical Isolates

1. Resistance Modifying Activity of Pomegranate
Fruit Constituents against
β-Lactamase Producing Gram-negative
Clinical Isolates
Ashok Laboratory Clinical Testing Centre Private Limited, Kolkata
Dr. Diganta Dey

2. Ciprofloxacin resistance: A challenge to antibiotic
treatment & infection control policies
Clinical isolates collected in our laboratory [2009–2010]
68% of the ESBL-producing Enterobacteriaceae
46% among the ESBL-non producer counterparts
Resistant to ciprofloxacin
Emerging resistance within extended-spectrum β-lactamase (ESBL)- &
metallo- β-lactamase (MBL)-producing Gram-negative bacilli
Increase in resistance to ciprofloxacin observed within <20 years of introduction
59.4% in China, 26% in Bangladesh, 14.7% in Spain
Astal, Singapore Med. J. 2005; Hooper, Emerg. Infect. Dis. 2001
Background...

3. Non-edible parts of pomegranate fruit:
To overcome ciprofloxacin resistance ??
Punica granatum Linn.
Edible fruit with nutritious & medicinal value
Phytochemical contents in pomegranate fruit extracts
Extract Phenolic Content
(mg GAE/g)
Flavonoid Content
(mg QE/g)
Juice of fruit (PGJ) 11.60 ± 0.3 0.06 ± 0.01
Methanol extract of pericarp (PGME) 229.23 ± 1.5 6.71 ± 0.01
Water extract of pericarp (PGWE) 158.1 ± 5.8 4.16 ± 0.01

4. Checkerboard protocol:
To check synergy of combination treatment in vitro
ANTIBIOTIC (e.g., ciprofloxacin)
Serially diluted in horizontal direction (1 to 12) of microtitre plate
RESISTANCE
MODIFIER
(e.g.,
PGME)
Serially
diluted
in
vertical
direction
(A
to
H)
of
plate
• 20 ESBL producing E. coli (viz., E1, E2, E3....E20)
• 20 ESBL producing K. pneumoniae (viz., K1, K2, K3....K20)
• 9 MBL producing P. aeruginosa (viz., P1, P2, P3....P9)
• studied against combination of pomegranate methanol extract
(PGME) and ciprofloxacin in vitro

5. Fluoroquinolone resistance percentage in
49 tesed ESBL and MBL producer isolates
[ Kirby and Bauer disc diffusion technique ]
0
10
20
30
40
50
60
70
80
90
100
Ciprofloxacin Ofloxacin Norfloxacin Levofloxacin
Resistance
(%)

6. Ciprofloxacin-PGME combination exhibited synergy (FIC
value for ciprofloxacin ≤0.5 was interpreted as ‘synergy’)
against:
• 40% of ESBL producer E. coli isolates
• 35% of ESBL producer K. pneumoniae isolates
• 44% of MBL producer P. aeruginosa isolates
Evaluation of synergy through isobologram

7. • The outcome of this study encouraged us to explore the active
ingredients of the fruit and to study their mechanism of action.
• Therefore, we tested the activity of some of its key
polyphenolic ingredients, namely caffeic acid, ellagic acid,
epigallocatechin-3-gallate (EGCG) and quercetin.
Conclusion...

8. The phytocompounds... And the antibiotics...
Ciprofloxacin
Gentamicin
Tetracycline
10 ESBL - producing K. pneumoniae isolates (viz., KpE-1, KpE-2,
KpE-3…KpE-10)
10 KPC-type carbapenemase - producing K. pneumoniae
isolates (viz., KpC-1, KpC-2, KpC-3…KpC-10)
The combination of these
3 antibiotics and
5 phytocompounds (including
reserpine as positive control)
was evaluated by the
checkerboard microtitre assay
against

9. Screening of the tested bacteria
(A) Double-disc synergy and (B) Phenotypic confirmatory test for ESBL detection.
(C) Modified Hodge Test of the four KPC producing K. pneumoniae isolates and K.
pneumoniae (ATCC 700603) as a negative control. All four KPC producing isolates
produced the characteristic cloverleaf-like indentation (indicated by a bracket),
except the negative control. Antibiotic discs used for these procedures were
(i) ceftazidime (30 μg), (ii) ceftazidime-clavulanic acid (30/10 μg), (iii) amoxy-
clavulanic acid (20/10 μg), (iv) cefotaxime (30 μg), and (v) imipenem (10 μg)

10. Interpretation of checkerboard data
FICI of ≤ 0.5: Synergism
FICI of 0.5 < to ≤ 1: Additive effect
FICI of 1 < to ≤ 2: Indifference
FICI of > 2: Antagonism
Mackay et al., Int J Antimicrob Agents, 2000

11. Activity of Caffeic acid
Combination Isolates* Number of isolates
Synergic
FICI ≤ 0.5
Additive
0.5 < FICI ≤ 1
Indifferent
1 < FICI ≤ 2
Caffeic acid +
Ciprofloxacin
S (6) 1 0 5
R-C (14) 7 1 6
Caffeic acid +
Gentamicin
S (16) 6 1 9
R-G (4) 2 0 2
Caffeic acid +
Tetracycline
S (12) 3 9 0
R-T (8) 4 1 3
*Number in parenthesis shows the number of isolates tested.
S: isolates sensitive to the tested antibiotic; R-C: ciprofloxacin-resistant
(MIC ≥ 4µg/ml) isolates; R-G: gentamicin-resistant (MIC ≥ 16 µg/ml); R-T:
tetracycline-resistant (MIC ≥ 16 µg/ml) isolates.

12. Activity of Ellagic acid
Combination Isolates* Number of isolates
Synergic
FICI ≤ 0.5
Additive
0.5 < FICI ≤ 1
Indifferent
1 < FICI ≤ 2
Ellagic acid +
Ciprofloxacin
S (6) 1 1 4
R-C (14) 1 9 4
Ellagic acid +
Gentamicin
S (16) 1 5 1
R-G (4) 1 1 2
Ellagic acid +
Tetracycline
S (12) 2 5 5
R-T (8) 3 2 3
*Number in parenthesis shows the number of isolates tested.
S: isolates sensitive to the tested antibiotic; R-C: ciprofloxacin-resistant
(MIC ≥ 4µg/ml) isolates; R-G: gentamicin-resistant (MIC ≥ 16 µg/ml); R-T:
tetracycline-resistant (MIC ≥ 16 µg/ml) isolates.

13. Activity of Epigallocatechin gallate (EGCG)
Combination Isolates* Number of isolates
Synergic
FICI ≤ 0.5
Additive
0.5 < FICI ≤ 1
Indifferent
1 < FICI ≤ 2
EGCG +
Ciprofloxacin
S (6) 4 2 0
R-C (14) 12 1 1
EGCG +
Gentamicin
S (16) 1 12 3
R-G (4) 1 2 1
EGCG +
Tetracycline
S (12) 11 1 0
R-T (8) 7 1 0
*Number in parenthesis shows the number of isolates tested.
S: isolates sensitive to the tested antibiotic; R-C: ciprofloxacin-resistant
(MIC ≥ 4µg/ml) isolates; R-G: gentamicin-resistant (MIC ≥ 16 µg/ml); R-T:
tetracycline-resistant (MIC ≥ 16 µg/ml) isolates.

14. Activity of Quercetin
Combination Isolates* Number of isolates
Synergic
FICI ≤ 0.5
Additive
0.5 < FICI ≤ 1
Indifferent
1 < FICI ≤ 2
Quercetin +
Ciprofloxacin
S (6) 1 3 2
R-C (14) 7 4 3
Quercetin +
Gentamicin
S (16) 5 6 5
R-G (4) 1 1 2
Quercetin +
Tetracycline
S (12) 0 8 4
R-T (8) 2 4 2
*Number in parenthesis shows the number of isolates tested.
S: isolates sensitive to the tested antibiotic; R-C: ciprofloxacin-resistant
(MIC ≥ 4µg/ml) isolates; R-G: gentamicin-resistant (MIC ≥ 16 µg/ml); R-T:
tetracycline-resistant (MIC ≥ 16 µg/ml) isolates.

15. Activity of Reserpine
(positive control of synergic interaction)
Combination Isolates* Number of isolates
Synergic
FICI ≤ 0.5
Additive
0.5 < FICI ≤ 1
Indifferent
1 < FICI ≤ 2
Reserpine +
Ciprofloxacin
S (6) 4 2 0
R-C (14) 13 1 0
Reserpine +
Gentamicin
S (16) 6 6 4
R-G (4) 1 2 1
Reserpine +
Tetracycline
S (12) 9 2 1
R-T (8) 6 2 0
*Number in parenthesis shows the number of isolates tested.
S: isolates sensitive to the tested antibiotic; R-C: ciprofloxacin-resistant
(MIC ≥ 4µg/ml) isolates; R-G: gentamicin-resistant (MIC ≥ 16 µg/ml); R-T:
tetracycline-resistant (MIC ≥ 16 µg/ml) isolates.

16. Time-kill curves
Time-kill curves of antimicrobials at one-quarter the MICs for
synergistic combinations
Ciprofloxacin and caffeic acid
against isolate # KpC-6
Ciprofloxacin and EGCG
against isolate # KpC-8
≥2log10 cfu/mL

17. Time-kill curves
Gentamicin and caffeic acid
against isolate # KpE-5
Gentamicin and quercetin
against isolate # KpE-4
Tetracycline and ellagic acid
against isolate # KpC-5
Tetracycline and EGCG
against isolate # KpE-9

18. Results
• The pure compounds exhibited synergy in
the range of 4 – 32 µg/ mL (data not shown)
• No antagonistic effect (FICI > 2) observed
• Synergic activity (FICI of ≤ 0.5) observed in
65% > 60% > 38.3% > 26.7% > 15% cases
when antibiotics tested in combination with
reserpine (positive control), EGCG, caffeic
acid, quercetin and ellagic acid, respectively.

19. Spectrofluorometric assay: EPI activity
of the phytochemicals

20. Transmission electron microscopy (TEM)
High-resolution TEM (HRTEM JEOL-TEM 2100, USA) analysis was
conducted to observe the morphology of ESBL-producer K.
pneumoniae (ATCC 700603) cells upon the treatment with 1 × MIC of
EGCG (128 µg/ml), 1 × MIC of tetracycline (16 µg/ml) and
combination of 0.5 × MIC EGCG and tetracycline (64 µg/ml + 8
µg/ml). Untreated bacterial cells were used as controls.

21. Transmission electron microscopy (TEM)

22. Transmission electron microscopy (TEM)

23. Transmission electron microscopy (TEM)

24. Conclusion
• It is probable that a fairly high content of
polyphenolics and flavonoids in PGME played
a crucial role in the inhibition of MDR efflux
• Subsequent study demonstrated prospective
EPI activity of two pomegranate fruit
constituents, viz., EGCG and caffeic acid,
against these bacteria.

25. Acknowledgement
• To my guides Dr. Banasri Hazra (Dept. of
Pharmaceutical Technology, Jadavpur University) and
Dr. Ratnamala Ray (Ashok Laboratory)
• To my friends Dr. Supratim Biswas (Dept. of
Metallurgical & Material Engineering, Jadavpur
University) and Mrs. Suparna Chakraborty ​(School of
Materials Science & Nanotechnology, Jadavpur
University)
• Technical and management supports provided by
Jadavpur University & Ashok Laboratory Clinical
Testing Centre Private Limited, Kolkata