Abstract of Doctoral Thesis - umfiasi.ro

Abstract of Doctoral Thesis

Doctoral Supervisor,

Prof. Dr. Anca MIRON

Doctoral Student,

Pharm. Sp. Petruța AELENEI

2020

ANTIBACTERIAL ACTIVITY OF SOME

PLANT EXTRACTS/COMPOUNDS –

INTERACTIONS WITH ANTIBIOTICS

Abstract of Doctoral Thesis

Doctoral Supervisor,

Prof. Dr. Anca MIRON

Doctoral Student,

Pharm. Sp. Petruța AELENEI

2020

1

TABLE OF CONTENTS

pg.

List of Abbreviations 4

GENERAL PART 5

CHAPTER 1. PLANT EXTRACTS/COMPOUNDS:

INTERACTIONS WITH

ANTIBIOTICS

5

1.1. Antibiotic resistance – overview 5

1.2. Synergy and tackling antibiotic resistance 9

1.3. Plant extracts/compounds tackling antibiotic

resistance

10

1.3.1. Plant extracts/compounds modulating

antibiotic activity against Staphylococcus

aureus

12


antibiotic activity against Staphylococcus

epidermidis

33


antibiotic activity against Escherichia coli

35


antibiotic activity against Pseudomonas

aeruginosa

43

PERSONAL PART 51

MOTIVATION AND AIMS OF DOCTORAL STUDY 51

CHAPTER 2. MATERIAL AND METHODS 53

2.1. Equipment 53

2.2. Solvents and reagents 53

2.3. Antibiotics 54

2.4. Culture media 54

2.5. Bacterial strains 54

2.6. Laboratory materials 55

2.7. Phytochemicals and plant extracts 55

2

2.8. Phytochemical studies 55

2.8.1. Chemical study of the essential oil isolated from

Coriandrum sativum L. (coriander) fruits

55

2.8.1.1. GC-MS qualitative chemical

analysis

55

2.8.1.2. GC-FID quantitative chemical

analysis

56

2.8.2. Chemical study of Morus alba L. (white

mulberry) leaf extract

56

2.8.2.1. HPLC-DAD-ESI-Q-TOF-MS/MS

qualitative chemical analysis

56

2.8.2.2. HPLC-DAD quantitative

chemical analysis

56

2.8.2.3. Quantification of total flavonoids 56

2.9. Evaluation of antibacterial activity 57

2.10. Evaluation of interactions between plant

extracts/compounds and antibiotics

58

2.10.1. Checkerboard assay 58

2.10.2. Time-kill assay 64

2.11. Evaluation of effects on bacterial membrane

integrity

67

2.11.1. Evaluation of bacterial membrane

permeabilization by fluorescence and

differential interference contrast

microscopy

67


morphology by scanning electron

microscopy

68

CHAPTER 3. RESULTS 69

3.1. Evaluation of bacterial susceptibility/resistance

to antibiotics

69

3.2. Chemical study of the essential oil isolated from

Coriandrum sativum L. (coriander) fruits by

gas chromatography – mass spectrometry and

gas chromatography with flame ionization

detection

70

3

3.3. Antibacterial activity of the essential oil isolated

from Coriandrum sativum L. (coriander) fruits

and linalool; interactions with antibiotics

73

3.4. Chemical study of Morus alba L. (white


83

3.4.1. Qualitative analysis by HPLC-DAD-ESI-Q-

TOF-MS/MS

83

3.4.2. Spectrophotometric quantification of total

flavonoids

90

3.4.3. Quantification of kuwanon G and morusin by

HPLC-DAD

90

3.5. Antibacterial activity of Morus alba L. (white

mulberry) leaf extract; interactions with

antibiotics

92

3.6. Antibacterial activity of morusin and kuwanon

G; interactions with antibiotics

95

3.6.1. Antibacterial activity of morusin and

kuwanon G against reference bacterial

strains

95


kuwanon G against bacterial strains isolated

from animals

96

3.6.3. Study of interactions between

morusin/kuwanon G and antibiotics

97

3.6.3.1. Study of interactions between

morusin/kuwanon G and

antibiotics against reference

Gram-positive bacterial strains

97

3.6.3.2. Study of interactions between

morusin/kuwanon G and

antibiotics against bacterial

strains isolated from animals

106



differential interference contrast microscopy

116


morphology by scanning electron

microscopy

118

4

3.7. Antibacterial activity of xanthohumol and 8-

prenylnaringenin; interactions with antibiotics

119

CHAPTER 4. DISCUSSIONS 130

4.1. Chemical study of the essential oil isolated from

Coriandrum sativum L. (coriander) fruits by

gas chromatography – mass spectrometry and

gas chromatography with flame ionization

detection

130

4.2. Antibacterial activity of the essential oil isolated

from Coriandrum sativum L. (coriander) fruits

and linalool; interactions with antibiotics

135

4.3. Chemical study of Morus alba L. (white


141

4.3.1. Qualitative analysis by HPLC-DAD-ESI-Q-

TOF-MS/MS

141

4.3.2. Quantification of total flavonoids, kuwanon

G and morusin

147


mulberry) ethanol leaf extract; interactions

with antibiotics

148

4.5. Antibacterial activity of morusin, kuwanon G,

xanthohumol and 8-prenylnaringenin;

interactions with antibiotics

156

GENERAL CONCLUSIONS. ORIGINAL

CONTRIBUTIONS. RESEARCH PERSPECTIVES

176

General conclusions 176

Original contributions 181

Research perspectives 183

REFERENCES 184

APPENDICES 209

5

LIST OF ABBREVIATIONS

ADN deoxyribonucleic acid

ARN ribonucleic acid

ATP adenosine triphosphate

CLSI Clinical and Laboratory Standards Institute

MIC minimum inhibitory concentration

DMSO dimethyl sulfoxide

EMA European Medicines Agency

ECDC European Centre for Disease Prevention and

Control

EUCAST European Committee on Antimicrobial

Susceptibility Testing

FDA Food and Drug Administration

FICI fractional inhibitory concentration index

GC-MS gas chromatography – mass spectrometry

GC-FID gas chromatography coupled with flame ionization

detection

HPLC high performance liquid chromatography

HPLC-DAD high performance liquid chromatography with

UV-VIS detection (diode array detector)

HPLC-

DAD-ESI-

Q-TOF-

MS/MS

high-performance liquid chromatography – diode

array detection – electrospray ionization –

quadrupole – time-of-flight – mass spectrometry

MRSA methicillin-resistant Staphylococcus aureus

MSSA methicillin-susceptible Staphylococcus aureus

SEM scanning electron microscopy

CFU colony forming units

UV-VIS ultraviolet – visible

VRSA vancomycin-resistant Staphylococcus aureus

6

Keywords

✓ Coriandrum sativum L.;

✓ linalool;

✓ Morus alba L.;

✓ prenylated flavonoids;

✓ synergism.

The Doctoral Thesis contains 208 pages, of which

45 belong to General Part and 157 belong to Personal

Part.

The Doctoral Thesis contains 51 figures, 44

tables, 5 appendices with 24 tables and 456 references.

In this Abstract, the selected figures, tables and

references have the numbers assigned in the Doctoral

Thesis.

7

ORIGINAL CONTRIBUTIONS

MOTIVATION AND AIMS OF THE DOCTORAL

STUDY

Although modern medicine has made huge

progress, bacterial infections, especially those caused by

antibiotic-resistant bacteria, represent a major global

public health problem. According to a World Health

Organization report in 2017, carbapenem-resistant A.

baumannii and P. aeruginosa, carbapenem-resistant and

extended spectrum beta-lactamase producing

Enterobacteriaceae are high risk pathogens belonging to

the critical-priority group. MRSA, vancomycin-resistant

E. faecium, fluoroquinolone-resistant Campylobacter spp.

and Salmonella spp., cephalosporin-resistant Neisseria

gonorrhoeae, clarithromycin-resistant H. pylori are high-

priority pathogens. These pathogens are multidrug-

resistant having the ability to transfer the antibiotic

resistance genes to other bacteria (199).

The development of novel antibiotics effective

against multidrug-resistant bacteria has become a global

priority. But over the past three decades, the number of

developed, approved and commercialized antibiotics

significantly decreased from 11 in 1980 – 1984 to only 6

in 2010 – 2014. This phenomenon has, in great part,

economic and legislative causes (200). In these

circumstances, the combination therapy (association of 2

or more antibiotics) was the main option for the treatment

of multidrug-resistant bacterial infections. Unfortunately,

8

combination antibiotic therapy was not always effective

(3, 201). In addition, antibiotic monotherapy and

combination antibiotic therapy cause side effects.

Antibiotics, even the usual ones, can cause numerous and

severe side effects (oxacillin: interstitial nephritis,

hepatitis; erythromycin: ototoxicity, cardiac side effects;

gentamicin: ototoxicity, nephrotoxicity; ciprofloxacin:

seizures, tendinitis, tendon rupture; tetracycline:

photosensitivity; clindamycin: gastrointestinal

dysfunctions, eosinophilia, maculopapular rash;

clarithromycin: encephalopathy; nitrofurantoin:

pulmonary infiltrate, acute respiratory insufficiency,

pulmonary fibrosis) (100, 202, 203).

Synergistic combinations of antibiotics with plant

extracts/compounds represent a promising therapeutic

alternative to antibiotic monotherapy and combination

antibiotic therapy. In recent years, numerous studies

reported the capacity of some plant extracts and

compounds (polyphenols, terpenoids, organosulfur

derivatives, alkaloids) to act synergistically with different

antibiotics, thus enhancing antibiotic efficacy against

pathogenic bacteria (including multidrug-resistant strains)

(204 – 208). Such synergistic interactions can be explained

by the capacity of plant metabolites to alter several

mechanisms involved in the antibiotic resistance of

pathogenic bacteria such as: modification in the target

sites of antibiotics, production of enzymes which

inactivate antibiotics, reduction in membrane permeability

and expression of efflux pumps (206).

Corilagin and tellimagrandin I reduce the synthesis

of penicillin-binding protein 2a (PBP2a) and its binding

capacity, thus increasing the activity of beta-lactam

9

antibiotics against MRSA (46, 206). PBP2a plays an

important role in the development of methicillin

resistance, enabling the synthesis of the bacterial cell wall

peptidoglycan in the presence of beta-lactam antibiotics.

Epigallocatechin gallate is an inhibitor of beta-lactamases,

enzymes which break the amidic bond in the beta-lactam

ring of penicillins and cephalosporins with their

inactivation (206). Eugenol, thymol and carvacrol increase

bacterial membrane permeability; a concentration of 1

mM of eugenol destroys 50% of the bacterial membrane

(159, 206, 209). The antibacterial effects of many essential

oils and polyphenolic extracts are due to the capacity of

volatile terpenoids and polyphenols to disrupt the bacterial

wall with leakage of cellular content (208, 209).

Numerous vegetal metabolites are efflux pumps

inhibitors. Carnosic acid and carnosol increase 2-4-fold

tetracycline activity against S. aureus via Tet (K) pump

inhibition. In addition, carnosic acid inhibits Msr (A)

pump, thus inducing 8-fold increase in erythromycin

activity against S. aureus. Baicalein acts synergistically

with ciprofloxacin against MRSA and gentamicin against

vancomycin-resistant enterococci, both synergisms being

ascribed to NorA efflux pump inhibition. Cinnamaldehyde

reduces clindamycin resistance of Clostridioides difficile

via CdeA efflux pump inhibition (206). Geraniol is

another efflux pump inhibitor; it potentiates the effects of

several antibiotics (ampicillin, penicillin, norfloxacin)

against multidrug-resistant Enterobacter aerogenes via

inhibition of AcrAB-TolC efflux system (165). Thymol

and carvacrol enhance tetracycline activity against S.

aureus via TetK pump inhibition (210).

10

Synergistic combinations of antibiotics with plant

extracts/compounds are promising for the development of

novel therapeutic strategies in bacterial infections.

The Doctoral Thesis aimed at investigating the

interactions which occur when combining plant

extracts/compounds with conventional antibiotics in order

to identify synergistic combinations which potentiate

antibiotic effects against pathogenic bacteria (including

antibiotic-resistant bacteria).

The main objectives of the Doctoral Thesis were:

− qualitative and quantitative chemical characterization

of plant extracts by spectrophotometric methods, gas

chromatography – mass spectrometry (GC-MS), gas

chromatography – flame ionization detection (GC-

FID), high-performance liquid chromatography –

diode array detection – electrospray ionization –

quadrupole – time-of-flight – mass spectrometry

(HPLC-DAD-ESI-Q-TOF-MS/MS), high-

performance liquid chromatography – diode array

detection (HPLC-DAD);

− evaluation of antibacterial activity of plant

extracts/compounds and conventional antibiotics

(broth microdilution method);

− evaluation of interactions between plant

extracts/compounds and conventional antibiotics

(checkerboard assay, time-kill assay);

− evaluation of effects of some plant compounds, alone

and combined with antibiotics, on MRSA membrane

integrity and morphology.

11

CAPITOLUL 3. RESULTS

3.4. Chemical study of Morus alba L. (white mulberry)

leaf extract

3.4.1. Qualitative analysis by HPLC-DAD-ESI-

Q-TOF-MS/MS

Fig. 3.7. HPLC-DAD chromatogram of Morus alba L. (white

mulberry) leaf extract (280 nm).

12

Tabel 3.V. Spectral and chromatographic data of constituents annotated in Morus alba L. (white mulberry) leaf

extract

No. Rt

(min)

[M-H]–

exp.

(m/z)

[M-H]–

calc. (m/z)

Error

(ppm) MF

ESI-MS/MS

fragment ions (m/z)

Proposed

identity

Phytochemical

class

1 29.8 567.1733 567.1719 2.41 C26H32O14 405, 243, 225, 199 Oxyresveratrol

dihexoside Stilbenoid

2 52.1 243.0670 243.0663 2.94 C14H12O4 225, 199 Oxyresveratrol Stilbenoid

3 63.6 241.0517 241.0506 4.56 C14H10O4 197, 119, 103 Moracin M 2-Arylbenzofuran

4 66.1 457.1513 457.1504 1.95 C24H26O9 324, 253, 211, 153, 103 Moracin P

pentoside 2-Arylbenzofuran

5 69.2 325.1084 325.1081 0.14 C19H18O5 253, 241, 225, 211,

109, 103 Moracin P 2-Arylbenzofuran

6 70.3 709.2281 709.2291 1.34 C40H38O12 599, 557, 489, 437,

371, 269 Moracenin D

Diels–Alder

adduct

7 71.5 579.1659 579.1661 0.27 C34H28O9

561, 469, 451, 359,

295, 269, 227, 175,

135, 109

Mulberrofuran C Diels–Alder

adduct

8 72.1 625.1723 625.1715 1.22 C35H30O11 499, 389, 353, 269,

227, 161, 125, 109 Kuwanon L

Diels–Alder

adduct

9 72.5 353.1048 353.1031 4.91 C20H18O6 297, 285, 231, 177,

151, 125, 109 Albanin A Prenylflavone

13


extract (cont.)

No. Rt

(min)

[M-H]–

exp.

(m/z)

[M-H]–

calc. (m/z)

Error

(ppm) MF

ESI-MS/MS

fragment ions (m/z)

Proposed

identity

Phytochemical

class

10 73.9 691.2138 691.2185 3.88 C40H36O11

673, 581, 539, 515,

471, 469, 419, 379,

353, 297, 271, 227,

161, 109

Kuwanon G a Diels–Alder

adduct

11 74.5 693.2323 693.2341 2.64 C40H38O11

675, 583, 567, 531,

473, 401; 353, 269,

227, 177

Kuwanon O Diels–Alder

adduct

12 75.1 351.0882 351.0874 2.24 C20H16O6 267, 177, 151, 125 Cyclocommunol Prenylflavone

13 75.3 437.1618 437.1606 2.79 C25H26O7

419, 393, 379, 297,

257, 217, 191, 173,

147, 125, 109

Morusinol Prenylflavone

14 75.8 759.2830 759.2811 2.52 C45H44O11

649, 581, 539, 471,

459, 405, 379, 353,

295, 227, 177

Kuwanon H Diels–Alder

adduct

15 77.0 761.2990 761.2967 2.97 C45H46O11 651, 581, 421, 353,

287, 259, 161, 125, 109 Sanggenol M

Diels–Alder

adduct

16

17

18

77.3

78.2

79.2

421.1652 421.1657 1.09 C25H26O6 309, 299, 297, 261, 231

Kuwanon C

Kuwanon A

Kuwanon F

Prenylflavone

14


extract (cont.)

No. Rt

(min)

[M-H]–

exp.

(m/z)

[M-H]–

calc. (m/z)

Error

(ppm) MF

ESI-MS/MS

fragment ions (m/z)

Proposed

identity

Phytochemical

class

19

20

80.8

82.1 419.1519 419.1500 4.49 C25H24O6

335, 309, 297, 217,

191, 121

Morusin a

Kuwanon B Prenylflavone

21 82.9 421.1673 421.1657 3.88 C25H26O6 309, 259, 231, 161, 103 Kuwanol C Prenylflavone

22 83.3 437.1618 437.1606 2.79 C25H26O7 419, 391, 379, 235,

177, 125 Hydroxymorusin Prenylflavone

23 85.0 391.1922 391.1915 1.83 C25H28O4 307, 293, 267, 253 Wittifuran B 2-Arylbenzofuran

24 89.3 417.1350 417.1344 1.53 C25H22O6 335, 265, 217, 173, 103 Cyclomorusin Prenylflavone

25 90.6 339.0885 339.0874 3.20 C19H16O6 255, 187,163 Moracin U 2-Arylbenzofuran

Legend: Rt – retention time, exp. – experimental, calc. – calculated, MF – molecular formula, [M-H]– pseudomolecular ion,

acompounds identified by comparison with retention time, UV and mass spectra of authentic standard

15


mulberry) leaf extract; interactions with

antibiotics

Fig. 3.15. Antibacterial activity of white mulberry leaf extract

against Gram-positive strains

Legend: MIC – minimum inhibitory concentration, ED – white mulberry leaf

extract, OX – oxacillin, AMX – amoxicillin, ERI – erythromycin, GEN –

gentamicin, CIP – ciprofloxacin, TE – tetracycline, CLI – clindamycin, ND –

not determined because inhibition of bacterial growth was not detected in the

tested concentration range

0

100

200

300

400

500

600

ED OX AMX ERI GEN CIP TE CLI

250

0.25 0.25 0.33 0.13 0.50 0.50 0.78

250

512

256

ND 8 0.5

128

ND

250

64 64

ND

256

0.5 1 ND

250

0.25

256

0.17 0.130.50

128

200

Th

e m

edia

n v

alu

es o

f M

IC (

µg

/mL

)

S. aureus ATCC 6538 S. aureus ATCC 33591

S. aureus ATCC 43300 S. epidermidis ATCC 12228

16

The study of the interactions between Morus alba L. (white mulberry) leaf extract and

antibiotics

Tabel 3.IX. Interactions between white mulberry leaf extract and antibiotics (checkerboard assay)

Agent MICAtb* DRIAtb MICED* DRIED FICI INT ΔE**

∑SYN (n) ∑ANT (n) INT

➢ S. aureus ATCC 33591

OX 128 4 7.81 32 0.28 S 1 (1) 0 (0) I

AMX 64 4 7.81 32 0.28 S 76 (6) 0 (0) I

GEN 0.25 32 62.50 4 0.28 S 1277.54 (26) 0 (0) S

CIP 0.25 2 125 2 1 Ad 0 (0) -51.71 (15) I

TE 64 2 7.81 32 0.53 Ad 0 (0) -194.23 (25) A


OX 2 32 62.50 4 0.28 S 0 (0) 67.82 (17) I

AMX 2 32 62.50 4 0.28 S 28 (8) 0 (0) I

GEN 64 4 7.81 32 0.28 S 969.00 (29) 0 (0) S

CIP 0.25 2 7.81 32 0.53 Ad 0 (0) -25.08 (27) I

TE 0.02 64 250 1 1.02 I 0 (0) -194.23 (25) A

17

Tabel 3.IX. Interactions between white mulberry leaf extract and antibiotics (checkerboard assay) (cont.)

Agent MICAtb* DRIAtb MICED* DRIED FICI INT ΔE**

∑SYN (n) ∑ANT (n) INT


OX 0.13 2 7.81 32 0.53 Ad 0.16 (1) 0 (0) I

AMX 0.13 2 7.81 32 0.53 Ad 96 (8) 0 (0) I

GEN 0.02 8 125 2 0.67 Ad 0 (0) -74.66 (26) I

CIP 0.25 2 7.81 32 0.53 Ad 0 (0) -62.66 (41) I

TE 0.50 1 7.81 32 1.03 I 0 (0) -66.88 (31) I

➢ S. epidermidis ATCC 12228

OX 0.13 2 7.81 32 0.53 Ad 0.16 (1) 0 (0) I

AMX 8 32 7.81 32 0.06 S 76 (10) 0 (0) I

GEN 0.02 8 7.81 32 0.16 S 1.13 (5) 0 (0) I

CIP 0.25 2 7.81 32 0.53 Ad 0 (0) -76.60 (15) I

TE 8 16 7.81 32 0.09 S 0 (0) -66.88 (31) I

Legend: *MIC – minimum inhibitory concentration in combination (µg/mL), ** n – number of combinations (amongst the 70

combinations for each strain) with statistically significant synergism (∑SYN) or antagonism (∑ANT), Atb – antibiotic, ED –

white mulberry leaf extract, OX – oxacillin, AMX – amoxicillin, ERI – erythromycin, GEN – gentamicin, CIP – ciprofloxacin,

TE – tetracycline, CLI – clindamycin, S – synergism, Ad – addition, I – indifference, A – antagonism, DRI – drug reduction

index, FICI – fractional inhibitory concentration index (median of three FICI calculated for three independent experimental

determinations), INT – interpretation

18

A

B

C

D

Fig. 3.16. Interactions between white mulberry leaf extract and antibiotics against S. aureus ATCC 33591 (A), S.

aureus ATCC 43300 (B), S. aureus ATCC 6538 (C) and S. epidermidis ATCC 12228 (D)

Legend: ED – white mulberry leaf extract, OX – oxacillin, AMX – amoxicillin, GEN – gentamicin, CIP – ciprofloxacin, TE –

tetracycline

0

0.5

1

0 0.5 1

FIC

ED

FICAntibiotic

S. aureus ATCC 33591

ED – OX ED – AMX ED – GEN ED – CIP ED – TE

0

0.5

1

0 0.5 1

FIC

ED

FICAntibiotic



0

0.5

1

0 0.5 1

FIC

ED

FICAntibiotic

S. aureus ATCC 6538


0

0.5

1

0 0.5 1

FIC

ED

FICAntibiotic

S. epidermidis ATCC 12228


19

3.6. Antibacterial activity of morusin and kuwanon G;

interactions with antibiotics


kuwanon G against reference bacterial

strains

Fig. 3.18. Antibacterial activity of morusin and kuwanon G against

Gram-positive strains

Legend: MIC – minimum inhibitory concentration, MO – morusin, KG –

kuwanon G, OX – oxacillin, AMX – amoxicillin, ERI – erythromycin, GEN

– gentamicin, CIP – ciprofloxacin, TE – tetracycline, CLI – clindamycin, ND

– not determined because inhibition of bacterial growth was not detected in

the tested concentration range

0

100

200

300

400

500

600

MO KG OX AMX ERI GEN CIP TE CLI

6.25 12.50 0.25 0.25 0.33 0.13 0.50 0.50 0.786.25 12.50

512

256

ND 8 0.50

128

ND6.25 12.50

64 64

ND

256

0.50 1 ND6.25 12.50

0.25

256

0.17 0.13 0.50

128

200

Th

e m

ed

ian

va

lues

of

MIC

(µ

g/m

L)

S. aureus ATCC 6538 S. aureus ATCC 33591

S. aureus ATCC 43300 S. epidermidis ATCC 12228

20

3.6.3. Study of interactions between morusin/kuwanon G and antibiotics

3.6.3.1. Study of interactions between morusin/kuwanon G and antibiotics

against reference Gram-positive bacterial strains

Tabel 3.X. Interactions between morusin and antibiotics against reference Gram-positive bacterial strains

Atb

Checkerboard assay Time-kill assay

MICAtb* DRIAtb MICMO* DRIMO FICI INT ΔE model*

Ma △LC24** INT ∑SYN (n) ∑ANT (n) INT


OX 16 32 0.78 8 0.16 S 3295.38 (67) 0 (0) S MO 2.04 ± 0.11 S

AMX 8 32 1.56 4 0.28 S 3282.81 (67) 0 (0) S MO 1.33 ± 0.32 Ad

GEN 0.13 64 0.78 8 0.14 S 3978.77 (51) 0 (0) S MO 1.50 ± 0.18 Ad

CIP 0.03 16 0.78 8 0.19 S 4285.14 (66) 0 (0) S CIP 2.02 ± 0.09 S

TE 16 8 0.78 8 0.25 S 3703.08 (62) 0 (0) S MO 1.17 ± 0.03 Ad

21

Tabel 3.X. Interactions between morusin and antibiotics against reference Gram-positive bacterial strains (cont.)

Atb





OX 1 64 0.78 8 0.14 S 3506.05 (69) 0 (0) S MO 2.61 ± 0.16 S

AMX 16 4 1.56 4 0.50 S 3822.01 (70) 0 (0) S MO 1.02 ± 0.50 Ad

GEN 16 16 0.39 16 0.13 S 4369.80 (60) 0 (0) S MO 2.15 ± 0.17 S

CIP 0.02 32 0.78 8 0.16 S 3654.80 (69) 0 (0) S CIP 1.18 ± 0.07 Ad

TE 0.02 64 0.78 8 0.14 S 3285.52 (70) 0 (0) S TE 1.49 ± 0.06 Ad


OX 0.02 16 1.56 4 0.31 S 3714.51 (68) 0 (0) S OX 1.56 ± 0.06 Ad

AMX 0.03 8 0.78 8 0.24 S 4807.48 (69) 0 (0) S AMX 1.03 ± 0.10 Ad

ERI 0.33 1 0.10 64 1.02 I 2964.55 (62) 0 (0) S ND

GEN 0.02 8 1.56 4 0.38 S 4037.23 (70) 0 (0) S GEN 1.56 ± 0.02 Ad

CIP 0.03 16 0.78 8 0.19 S 3903.85 (70) 0 (0) S CIP 1.05 ± 0.03 Ad

TE 0.02 32 1.56 4 0.28 S 3271.33 (68) 0 (0) S TE 1.56 ± 0.32 Ad

CLI 0.01 128 3.13 2 0.51 Ad 0 (0) -779.17 (61) A ND

22

Tabel 3.X. Interactions between morusin and antibiotics against reference Gram-positive bacterial strains (cont.)

Atb





OX 0.02 16 1.56 4 0.31 S 4132.73 (63) 0 (0) S OX 1.80 ± 0.18 Ad

AMX 16 16 0.10 64 0.08 S 2487.15 (61) 0 (0) S MO 1.47 ± 0.06 Ad

ERI 0.01 16 0.78 8 0.18 S 3882.77 (59) 0 (0) S ERI 1.03 ± 0.06 Ad

GEN 0.02 8 1.56 4 0.38 S 4511.68 (68) 0 (0) S GEN 1.24 ± 0.09 Ad

CIP 0.02 32 1.56 4 0.28 S 3953.39 (66) 0 (0) S CIP 1.54 ± 0.23 Ad

TE 0.25 512 1.56 4 0.25 S 4124.92 (66) 0 (0) S MO 1.29 ± 0.06 Ad

CLI 50 4 0.10 64 0.27 S 3575.29 (70) 0 (0) S MO 0.25 ± 0.07 I

Legend: *MIC – minimum inhibitory concentration in combination (µg/mL), **n – number of combinations (amongst the 70

combinations for each strain) with statistically significant synergism (∑SYN) or antagonism (∑ANT), ***the increase in bacterial

killing induced by the combination in comparison with its most active component tested alone (expressed in log10 CFU/mL at

24 h incubation, mean ± standard deviation), Atb – antibiotic, MO – morusin, OX – oxacillin, AMX – amoxicillin, ERI –

erythromycin, GEN – gentamicin, CIP – ciprofloxacin, TE – tetracycline CLI – clindamycin, S – synergism, Ad – addition, I –

indifference, A – antagonism, DRI – dose reduction index, FICI – fractional inhibitory concentration index (median of three FICI

calculated for three independent experimental determinations), INT – interpretation, Ma – most active agent of combination, ND – not

determined

23

A.

E.

B.

F.

Fig. 3.20. Interactions between morusin/kuwanon G and antibiotics against S. aureus ATCC 33591 (A, E), S.

aureus ATCC 43300 (B, F), S. aureus ATCC 6538 (C, G), S. epidermidis ATCC 12228 (D, H)

0

0.5

1

0 0.5 1

FIC

MO

FICAntibiotic


MO – OX MO – AMX MO – GEN

MO – CIP MO – TE

0

0.5

1

0 0.5 1

FIC

KG

FICAntibiotic


KG – OX KG – AMX KG – GEN KG – CIP KG – TE

0

0.5

1

0 0.5 1

FIC

MO

FICAntibiotic


MO – OX MO – AMX MO – GEN

MO – CIP MO – TE

0

0.5

1

0 0.5 1

FIC

KG

FICAntibiotic


KG – OX KG – AMX KG – GEN

KG – CIP KG – TE

24

C.

G.

D.

H.

Fig. 3.20. Interactions between morusin/kuwanon G and antibiotics against S. aureus ATCC 33591 (A, E), S.

aureus ATCC 43300 (B, F), S. aureus ATCC 6538 (C, G), S. epidermidis ATCC 12228 (D, H) (cont.)

Legend: FIC – fractional inhibitory concentration, MO – morusin, KG – kuwanon G, OX – oxacillin, AMX – amoxicillin,

GEN – gentamicin, CIP – ciprofloxacin, TE – tetracycline, ERI – erythromycin

0

0.5

1

0 0.5 1

FIC

MO

FICAntibiotic

S. aureus ATCC 6538

MO – OX MO – AMX MO – GEN MO – CIP

MO – TE MO – ERI MO – CLI

0

0.5

1

0 0.5 1

FIC

KG

FICAntibiotic

S. aureus ATCC 6538

KG – OX KG – AMX KG – GEN KG – CIP

KG – TE KG – ERI KG – CLI

0

0.5

1

0 0.5 1

FIC

MO

FICAntibiotic


MO – OX MO – AMX MO – GEN MO – CIP

MO – TE MO – ERI MO – CLI

0

0.5

1

0 0.5 1

FIC

KG

FICAntibiotic


KG – OX KG – AMX KG – GEN KG – CIP

KG – TE KG – ERI KG – CLI

25

A.

B.

Fig. 3.23. Time-kill curves revealing synergistic interactions between morusin/kuwanon G and antibiotics

against S. aureus ATCC 33591 (A, C, E, F) and S. aureus ATCC 43300 (B, D)

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40 44 48

log

10 C

FU

/mL

Time (h)


Bacterial growth control

Oxacillin – ½ CMI (256 μg/mL)

Morusin – ½ CMI (3.13 μg/mL)

Morusin – ½ CMI (3.13 μg/mL) + Oxacillin

– ½ CMI (256 μg/mL)

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40 44 48

log

10 C

FU

/mL

Time (h)





Morusin – ½ CMI (3.13 μg/mL) + Oxacillin

– ½ CMI (32 μg/mL)

26

C.

D.


against S. aureus ATCC 33591 (A, C, E, F) and S. aureus ATCC 43300 (B, D) (cont.)

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40 44 48

log

10 C

FU

/mL

Time (h)



Ciprofloxacin – ½ CMI (0.25 μg/mL)


Morusin – ½ CMI (3.13 μg/mL) + Ciprofloxacin

– ½ CMI (0.25 μg/mL)

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40 44 48

log

10 C

FU

/mL

Time (h)



Gentamicin – ½ CMI (128 μg/mL)


Morusin – ½ CMI (3.13 μg/mL) + Gentamicin

– ½ CMI (128 μg/mL)

27

E.

F.


against S. aureus ATCC 33591 (A, C, E, F) and S. aureus ATCC 43300 (B, D) (cont.)

Legend: MIC – minimum inhibitory concentration

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40 44 48

log

10 C

FU

/mL

Time (h)




Kuwanon G – ½ CMI (6.25 μg/mL)

Kuwanon G – ½ CMI (6.25 μg/mL) + Oxacillin

– ½ CMI (256 μg/mL)

0

2

4

6

8

10

0 4 8 12 16 20 24 28 32 36 40 44 48

log

10 C

FU

/mL

Time (h)



Ciprofloxacin – ½ CMI (0.25 μg/mL)

Kuwanon G – ½ CMI (6.25 μg/mL)

Kuwanon G – ½ CMI (6.25 μg/mL) + Ciprofloxacin

– ½ CMI (0.25 μg/mL)

28

Tabel 3.XI. Interactions between kuwanon G and antibiotics against reference Gram-positive bacterial strains

Atb


MICAtb* DRIAtb MICKG* DRIKG FICI INT ΔE model**

Ma △LC24*** INT ∑SYN (n) ∑ANT (n) INT


OX 128 4 3.13 4 0.50 S 0 (0) -24.54 (10) I KG 2.70 ± 0.61 S

AMX 16 16 3.13 4 0.31 S 1983.36 (31) 0 (0) S KG 0.89 ± 0.10 I

GEN 1 8 0.20 64 0.14 S 3477.78 (64) 0 (0) S GEN 0.67 ± 0.06 I

CIP 0.13 4 0.39 32 0.28 S 3602.06 (60) 0 (0) S CIP 2.63 ± 0.21 S

TE 0.25 512 6.25 2 0.50 S 4000.00(15) 0 (0) S KG 0.18 ± 0.03 I


OX 1 64 3.13 4 0.27 S 442.20 (47) 0 (0) S KG 1.08 ± 0.01 Ad

AMX 16 4 3.13 4 0.50 S 442.20 (0) 0 (0) S KG 0.14 ± 0.06 I

GEN 32 8 0.78 16 0.19 S 0 (0) 0 (0) I KG 0.77 ± 0.05 I

CIP 0.02 32 3.13 4 0.38 S 3385.19 (69) 0 (0) S CIP 1.72 ± 0.03 Ad

TE 0.25 4 1.56 8 0.38 S 1492.86 (37) 0 (0) S KG 0.18 ± 0.03 I

29

Tabel 3.XI. Interactions between kuwanon G and antibiotics against reference Gram-positive bacterial strains (cont.)

Atb


MICAtb* DRIAtb MICKG* DRIKG FICI INT ΔE model**

Ma △LC24*** INT ∑SYN (n) ∑ANT (n) INT


OX 0.06 4 0.20 64 0.27 S 0 (0) -328.7 (31) A OX 0.67 ± 0.06 I

AMX 0.25 1 0.20 64 1.02 I 1360.55 (41) 0 (0) S ND

ERI 0.17 2 1.56 8 0.63 Ad 0 (0) 0 (0) I ND

GEN 0.03 4 12.50 2 0.75 Ad 1526 (36) 0 (0) S ND

CIP 0.13 4 0.20 64 0.27 S 3442.77 (59) 0 (0) S CIP 0.56 ± 0.09 I

TE 0.25 2 12.50 2 1 Ad 1416.67 (18) 0 (0) S ND

CLI 0.01 128 12.50 1 1.01 I 3575.29 (70) 0 (0) S ND


OX 0.13 2 0.20 64 0.52 Ad 0 (0) -10.74 (2) I ND

AMX 16 16 0.40 32 0.09 S 2144.58 (45) 0 (0) S AMX 0.53 ± 0.06 I

ERI 0.01 16 6.25 2 0.56 Ad 81.33 (19) 0 (0) I ND

GEN 0.02 8 3.13 4 0.38 S 2284 (57) 0 (0) S GEN 0.90 ± 0.07 I

CIP 0.13 4 0.20 64 0.28 S 3547.84 (59) 0 (0) S CIP 1.56 ± 0.06 Ad

TE 64 2 1.56 8 0.62 Ad 2455 (24) 0 (0) S ND

CLI 1 200 12.50 1 1.01 I 5.30 (5) 0 (0) I KG 0.23 ± 0.08 I

Legend: *MIC – minimum inhibitory concentration in combination (µg/mL), **n – number of combinations (amongst the 70 combinations for each strain)

with statistically significant synergism (∑SYN) or antagonism (∑ANT), ***the increase in bacterial killing induced by the combination in comparison with

its most active component tested alone (expressed in log10 CFU/mL at 24 h incubation, mean ± standard deviation), Atb – antibiotic, KG – kuwanon G, OX – oxacillin, AMX – amoxicillin, ERI – erythromycin, GEN – gentamicin, CIP – ciprofloxacin, TE – tetracycline CLI – clindamycin, S – synergism, Ad –

addition, I – indifference, A – antagonism, DRI – dose reduction index, FICI – fractional inhibitory concentration index (median of three FICI calculated for

three independent experimental determinations), INT – interpretation, Ma – most active agent of combination, ND – not determined

30



differential interference contrast

microscopy

Fig. 3.27. Effects of exposure to morusin (2 × MIC) and kuwanon G

(2 × MIC) on membrane permeability of exponential-phase cultures

of S. aureus ATCC 43300 (MRSA) expressed as the percentage of

red fluorescent cells in the population (each bar represents the mean

acquired by counting the captured cells and the error bars represent

the standard deviations) Legend: MIC – minimum inhibitory concentration

3.000 0 0.71 0

40.55

68.19

94.4796.91 98.20

40.41 40.09

90.03 91.18

98.37

0

10

20

30

40

50

60

70

80

90

100

15 30 60 120 180

red f

luore

scen

t ce

lls

(%)

Time (minutes)

Control

Morusin 12.50 μg/mL (2 × MIC)

Kuwanon G 25 μg/mL (2 × MIC)

31

A

B

C

D

E

F

Fig. 3.28. Fluorescent images of S. aureus ATCC 43300 (MRSA) after 60 minutes of exposure at DMSO

(control) (A, D), morusin (B, E) and kuwanon G (C, F) by fluorescence microscopy (A, B, C) and differential

phase contrast microscopy (D, E, F)

32

3.6.5. Evaluation of bacterial membrane morphology by scanning electron microscopy

Fig. 3.29. Scanning electronic microscopy of S. aureus ATCC 43300 (MRSA) after 24 h incubation at 37°C with

no treatment (control) (A) and treated with oxacillin (1/2 × MIC) (B), morusin (1/2 × MIC) (C), morusin (1/2 ×

MIC) and oxacillin (1/2 × MIC) (D), kuwanon G (1/2 × MIC) (E) and kuwanonă G (1/2 × MIC) and oxacillin

(1/2 × MIC) (F) Legend: MIC – minimum inhibitory concentration, MO – morusin, KG – kuwanon G, OX – oxacillin

33

GENERAL CONCLUSIONS. ORIGINAL

CONTRIBUTIONS. RESEARCH PERSPECTIVES

GENERAL CONCLUSIONS

The current Doctoral Thesis investigated the

interactions between plant extracts/compounds and

antibiotics in order to identify synergistic combinations in

which the antibiotic effects against pathogenic bacteria,

including antibiotic-resistant bacteria are potentiated. The

plant extracts included in this study were chemically

characterized by performant hyphenated methods to

determine correlations between the chemical composition

and antibacterial activity. The capacity of some plant

compounds/plant compound-antibiotic combinations to

alter the integrity and morphology of bacterial membrane

was also investigated.

The research studies led to the following

conclusions:

GC-MS and GC-FID analyses of the essential oil

isolated from the fruits of Coriandrum sativum L.

(coriander) identified linalool as the major constituent

(70.11%); other constituents of coriander essential oil: -

pinen (5.46%), camphor (4.96%), γ-terpinen (4.37%),

geranyl acetate (3.51%), D-limonen (2.49%) and trans-

geraniol (1.86%).

34

There were identified bacteriostatic synergistic

combinations between coriander fruit essential oil and

the following antibiotics:

− oxacillin – against S. aureus ATCC 33591 (MRSA) (8-

fold reduction in MICoxacillin in combination);

− amoxicillin – against S. aureus ATCC 33591 and

ATCC 43300 (both MRSA) and S. epidermidis ATCC

12228 (16, 4 and 32-fold reduction in MICamoxicillin in

combinations, respectively);

− gentamicin – against S. aureus ATCC 33591 and

ATCC 43300 (both MRSA), S. aureus ATCC 6538

(MSSA), S. epidermidis ATCC 12228, P. aeruginosa

ATCC 9027 and ATCC 27853 (8, 16, 4, 8, 64 and 64-

fold reduction in MICgentamicin in combinations,

respectively);

− erythromycin – against P. aeruginosa ATCC 9027 and

ATCC 27853, E. coli ATCC 25922 (16-fold reduction

in MICerythromycin in combinations);

− ciprofloxacin – against S. epidermidis ATCC 12228,

P. aeruginosa ATCC 27853 (4-fold reduction in

MICciprofloxacin in combinations);

− tetracycline – against S. aureus ATCC 33591

(MRSA), S. aureus ATCC 6538 (MSSA), S.

epidermidis ATCC 12228, P. aeruginosa ATCC

27853 and E. coli ATCC 25922 (8, 4, 512, 4 and 8-

fold reduction in MICtetracycline in combinations,

respectively).

5 of these combinations (coriander essential oil –

oxacillin against S. aureus ATCC 33591 (MRSA),

coriander essential oil – amoxicillin against S. aureus

ATCC 33591 and ATCC 43300 (both MRSA) and S.

epidermidis ATCC 12228, coriander essential oil –

35

tetracycline against S. epidermidis ATCC 12228) were

confirmed to be synergistic by E model too. On the other

hand, E model found the combinations coriander

essential oil – oxacillin to be synergistic against S. aureus

ATCC 43300 (MRSA), S. aureus ATCC 6538 (MSSA)

and S. epidermidis ATCC 12228; these combinations were

identified as additive and indifferent on the basis of FICI

values.


combinations between linalool and the following

antibiotics:

− oxacillin – against S. aureus ATCC 33591 and ATCC

43300 (both MRSA) (32 and 4-fold reduction in

MICoxacillin in combinations, respectively);







12228 (8-fold reduction in MICgentamicin in

combinations);

− erythromycin – against S. aureus ATCC 6538 (MSSA)

and E. coli ATCC 25922 (4-fold reduction in

MICerythromycin in combinations);

− ciprofloxacin – against S. aureus ATCC 33591 and


(MSSA) and S. epidermidis ATCC 12228 (4-fold

reduction in MICciprofloxacin in combinations);

− tetracycline – against S. aureus ATCC 33591 and

ATCC 43300 (both MRSA), S. epidermidis ATCC

36

12228, P. aeruginosa ATCC 27853 and E. coli ATCC

25922 (4, 4, 16, 8 and 256-fold reduction in

MICtetracycline in combinations, respectively).

3 of these combinations (linalool – amoxicillin

against S. aureus ATCC 33591 and ATCC 43300 (both

MRSA), linalool – tetracycline against S. epidermidis

ATCC 12228) were confirmed to be synergistic by E

model too. The E model found the combination linalool

– gentamicin to be synergistic against S. aureus ATCC

6538 (MSSA), this combination being identified as

additive on the basis of FICI value.

HPLC-DAD-ESI-Q-TOF-MS/MS analysis of

Morus alba L. leaf extract led to the annotation of 25

constituents: 2 stilbene derivatives (oxyresveratrol and

oxyresveratrol dihexoside), 5 2-arylbenzofurans

(moracin M, moracin P and moracin P pentoside, moracin

U, wittifuran B), 7 Diels–Alder type adducts (moracenin

D, mmulberrofuran C, kuwanon G, H, L and O, sanggenol

M), 11 prenylflavonoids (albanin A, cyclocommunol,

morusinol, kuwanon A, B, C and F, kuwanol C, morusin,

hydroxymorusin, cyclomorusin).

Quantitative chemical analysis of Morus alba L.

leaf extract showed a total flavonoid content of 57.83 ±

4.64 mg/g extract, 10.47 ± 0.13 mg morusin/g extract

and 15.42 ± 0.25 mg kuwanon G/g extract.


combinations between white mulberry leaf extract and

the following antibiotics:

37

− oxacillin – against S. aureus ATCC 33591 and S.

aureus ATCC 43300 (both MRSA) (4 and 32-fold

reduction in MICoxacillin in combinations, respectively);







12228 (32, 4 and 8-fold reduction in MICgentamicin in


− tetracycline – against S. epidermidis ATCC 12228 (16-

fold reduction in MICtetracycline in combination).

Among these combinations, only the combinations

white mulberry leaf extract – gentamicin against both

MRSA strains (S. aureus ATCC 33591 and ATCC 43300)

were confirmed to be synergistic by E model too.


combinations between morusin and the following

antibiotics:


aureus ATCC 43300 (both MRSA), S. aureus ATCC

6538 (MSSA) and S. epidermidis ATCC 12228 (32,

64, 16 and 16-fold reduction in MICoxacillin in




and S. epidermidis ATCC 12228 (32, 4, 8 and 16-fold

reduction in MICamoxicillin in combinations,

respectively);

38



(MSSA), S. epidermidis ATCC 12228 (64, 16, 8 and

8-fold reduction in MICgentamicin in combinations,

respectively);

− erythromycin – against S. epidermidis ATCC 12228

(16-fold reduction in MICerythromycin in combination);



(MSSA) and S. epidermidis ATCC 12228 (16, 32, 16

and 32-fold reduction in MICciprofloxacin in


− tetracycline – against S. aureus ATCC 33591 and S.

aureus ATCC 43300 (both MRSA), S. aureus ATCC

6538 (MSSA) and S. epidermidis ATCC 12228 (8, 64,

32 and 512-fold reduction in MICtetracycline in


− clindamycin – against S. epidermidis ATCC 12228 (4-

fold reduction in MICclindamycin in combination).

All these synergisms were also confirmed by E

model. In addition, E model also identified the

combination morusin – erythromycin against S. aureus

ATCC 6538 (MSSA) as synergistic, this combination

being identified as indifferent based on the FICI value.

There were identified bactericidal synergistic

combinations between morusin (½ MIC) and the

following antibiotics (½ MIC):


43300 (both MRSA);

39

− ciprofloxacin – against S. aureus ATCC 33591

(MRSA);

− gentamicin – against S. aureus ATCC 43300

(MRSA).

Morusin (2 MIC) increased the permeability

S. aureus ATCC 43300 (MRSA) membrane (40.55%

after 15 min. exposure, 94.47% after 60 min. exposure,

98.20% after 180 min. exposure).

Morusin (½ MIC) and the combination

morusin – oxacillin (both at ½ MIC) led to leakage of

cellular content in S. aureus ATCC 43300 (MRSA).

Morusin acted synergistically with various

antibiotics (oxacillin, amoxicillin, gentamicin,

ciprofloxacin, tetracycline, clindamycin) against

oxacillin-resistant MRSA strains isolated from

animals.


combinations between kuwanon G and the following

antibiotics:


aureus ATCC 43300 (both MRSA) and S. aureus

ATCC 6538 (MSSA) (4, 64 and 4-fold reduction in

MICoxacillin in combinations, respectively);





40



12228 (8-fold reduction in MICgentamicin in

combinations);



(MSSA) and S. epidermidis ATCC 12228 (4, 32, 4 and

4-fold reduction in MICciprofloxacin in combinations,

respectively);

− tetracycline – against S. aureus ATCC 33591 and S.

aureus ATCC 43300 (both MRSA) (512 and 4-fold

reduction in MICtetracycline in combinations,

respectively);

3 of these combinations (kuwanon G – oxacillin

against S. aureus ATCC 33591 (MRSA) and S. aureus

ATCC 6538 (MSSA), kuwanon G – gentamicin against S.

aureus ATCC 43300 (MRSA)) were not confirmed to be

synergistic by E model. On the other hand, some additive

and indifferent combinations according to FICI values

(kuwanon G – amoxicillin, kuwanon G – gentamicin,

kuwanon G – tetracycline and kuwanon G – clindamycin

against S. aureus ATCC 6538 (MSSA), kuwanon G –

tetracycline against S. epidermidis ATCC 12228) were

identified as synergistic on the basis of E model.


combinations between kuwanon G (½ MIC) and

oxacillin and ciprofloxacin (both at ½ MIC) against S.

aureus ATCC 33591 (MRSA).

41

Kuwanon G (2 MIC) increased the

permeability of S. aureus ATCC 43300 (MRSA)

membrane (40.41% after 15 min. exposure, 90.03% after

60 min. exposure, 98.3% after 180 min. exposure).

Kuwanon G (½ MIC) and the combination

kuwanon G – oxacillin (both at ½ MIC) led to leakage

of cellular content in S. aureus ATCC 43300 (MRSA).

Kuwanon G acted synergistically with various

antibiotics (oxacillin, amoxicillin, gentamicin,

ciprofloxacin, tetracycline) against oxacillin-resistant

MRSA strains isolated from animals.


combinations between xanthohumol and the following

antibiotics:


43300 (both MRSA) and S. aureus ATCC 6538

(MSSA) (256, 64 and 4-fold reduction in MICoxacillin in



ATCC 43300 (both MRSA) S. aureus ATCC 6538


32-fold reduction in MICamoxicillin in combinations,

respectively);





respectively);

42


(MRSA) and S. aureus ATCC 6538 (MSSA) (4-fold

reduction in MICciprofloxacin in combinations);

− erythromycin – against S. epidermidis ATCC 12228

(32-fold reduction in MICerythromycin in combination);

− clindamycin – against S. epidermidis ATCC 12228 (4-

fold reduction in MICclindamycin in combination).

Among all these synergistic combinations, E

model confirmed only the combinations xanthohumol -

gentamicin against all 4 Gram-positive strains,

xanthohumol - ciprofloxacin against S. aureus ATCC

43300 (MRSA) and S. aureus ATCC 6538 (MSSA) and

xanthohumol - amoxicillin against S. epidermidis ATCC

12228.


combinations between xanthohumol (½ MIC) and the

following antibiotics (½ MIC):


ATCC 43300 (both MRSA);


(MRSA).


combinations between 8-prenylnarigenin and the

following antibiotics:


43300 (both MRSA), S. aureus ATCC 6538 (MSSA)

and S. epidermidis ATCC 12228 (4, 64, 4 and 4-fold

reduction in MICoxacillin in combinations, respectively);

43





respectively);


ATCC 43300 (both MRSA) and S. aureus ATCC 6538

(MSSA) (4-fold reduction in MICciprofloxacin in

combinations);

− tetracycline – against S. aureus ATCC 6538 (MSSA)

and S. epidermidis ATCC 12228 (4 and 16-fold

reduction in MICtetracycline in combinations,

respectively);

− erythromycin – against S. aureus ATCC 6538 (MSSA)

(8-fold reduction in MICerythromycin in combination).

Among all these synergistic combinations, E

model confirmed only the combinations 8-prenylnarigenin

- gentamicin against all 4 Gram-positive strains, 8-

prenylnarigenin - ciprofloxacin against S. aureus ATCC

33591 (MRSA) and S. aureus ATCC 6538 (MSSA) and 8-

prenylnarigenin - oxacillin against S. epidermidis ATCC

12228.


combinations between 8-prenylnarigenin (½ MIC)

and the following antibiotics (½ MIC):





44


(MSSA).

45

ORIGINAL CONTRIBUTIONS

The originality of the Doctoral Thesis is supported

by:

✓ identification, on the basis of FICI values, of 12

combinations coriander fruit essential oil – antibiotics

exhibiting synergistic bacteriostatic effects against

Gram-positive bacteria: S. aureus ATCC 33591 and S.

aureus ATCC 43300 (both MRSA) (6 combinations),

S. aureus ATCC 6538 (MSSA) (2 combinations) and S.

epidermidis ATCC 12228 (4 combinations). Among

these combinations, 5 synergistic combinations were

also confirmed by E model;


combinations coriander fruit essential oil – antibiotics


Gram-negative bacteria: P. aeruginosa ATCC 9027 (2

combinations) and ATCC 27853 (4 combinations) and

E. coli ATCC 25922 (2 combinations);


combinations linalool – antibiotics exhibiting

synergistic bacteriostatic effects against Gram-positive

bacteria: S. aureus ATCC 33591 and ATCC 43300

(both MRSA) (10 combinations), S. aureus ATCC

6538 (MSSA) (2 combinations) and S. epidermidis

ATCC 12228 (4 combinations). Among these

combinations, 3 synergistic combinations were also

confirmed by E model;


combinations linalool – antibiotics exhibiting

synergistic bacteriostatic effects against Gram-negative

46

bacteria: P. aeruginosa ATCC 27853 (one

combination) and E. coli ATCC 25922 (2

combinations);


combinations white mulberry leaf extract – antibiotics



aureus ATCC 43300 (both MRSA) (6 combinations)

and S. epidermidis ATCC 12228 (3 combinations).

Among these combinations, 2 synergistic combinations

were also confirmed by E model;


combinations morusin – antibiotics exhibiting


bacteria: S. aureus ATCC 33591 and S. aureus ATCC

43300 (both MRSA) (10 combinations), S. aureus

ATCC 6538 (MSSA) (5 combinations) and S.

epidermidis ATCC 12228 (7 combinations). All these

synergistic combinations were also confirmed by E

model;

✓ identification, on the basis of △LC24 values, of 4


synergistic bactericidal effects against S. aureus ATCC

33591 and S. aureus ATCC 43300 (both MRSA);



synergistic bacteriostatic effects against 4 MRSA

strains resistant to oxacillin, isolated from animals.

Among these, 13 synergistic combinations were also




47

synergistic bactericidal effects against MRSA strains

isolated from animals;


combinations kuwanon G – antibiotics exhibiting


bacteria: S. aureus ATCC 33591 and S. aureus ATCC

43300 (both MRSA) (10 combinations), S. aureus

ATCC 6538 (MSSA) (2 combinations) and S.







33591 (MRSA);



synergistic bacteriostatic effects against 4 MRSA

strains resistant to oxacillin, isolated from animals.

All these synergistic combinations were also




synergistic bacteriostatic effects against MRSA strains

isolated from animals;

✓ revealing the ability of morusin, kuwanon G and their

combinations with oxacillin to damage MRSA

membrane;


combinations xanthohumol – antibiotics exhibiting


bacteria: S. aureus ATCC 33591 and ATCC 43300

48

(both MRSA) (7 combinations), S. aureus ATCC 6538

(MSSA) (4 combinations) and S. epidermidis ATCC

12228 (4 combinations). Among these combinations, 7

synergistic combinations were also confirmed by E

model;


combinations xanthohumol – antibiotics exhibiting


33591 and S. aureus ATCC 43300 (both MRSA);


combinations 8-prenylnaringenin – antibiotics



aureus ATCC 43300 (both MRSA) (6 combinations),

S. aureus ATCC 6538 (MSSA) (5 combinations) and S.





combinations 8-prenylnaringenin – antibiotics

exhibiting synergistic bactericidal effects against S.

aureus ATCC 33591 and S. aureus ATCC 43300 (both

MRSA) and S. aureus ATCC 6538 (MSSA).

49

RESEARCH PERSPECTIVES

The results obtained in the current Doctoral Thesis

support future research directions:

✓ investigation of the antibacterial potential against

other Gram-positive and Gram-negative bacteria,

including multidrug-resistant clinical isolates;

✓ in vivo assessment of antibacterial potential of

synergistic combinations (animal models);

✓ in vivo assessment of toxicological profile of

synergistic combinations (animal models);

✓ incorporation of plant extract/compound in a

formulation to increase its oral bioavailability;

✓ clinical assessment of the efficacy and safety of

synergistic combinations plant extract/compound –

antibiotic.

50

REFERENCES (SELECTION)

3. Cheesman MJ, Ilanko A, Blonk B, Cock IE.

Developing New Antimicrobial Therapies: Are

Synergistic Combinations of Plant

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