Preliminary Study on the Antibacterial Activity of Essential Oils Alone and in Combination with Gentamicin Against Extended-Spectrum β-Lactamase-Producing and New Delhi Metallo-β-Lactamase-1-Producing Klebsiella pneumoniae Isolates

Abstract

Aim:

The aim of the study was to investigate possible synergistic effects between several selected, commercially available essential oils and gentamicin against extended-spectrum β-lactamase (ESBL)-producing and New Delhi metallo-β-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae isolates.

Materials and Methods:

ESBLs production was confirmed by double-disk synergy test. Isolates positive for bla_NDM-1 gene were found among the tested strains. K. pneumoniae ATCC^® BAA-1705™ strain was used as a control. The checkerboard method was applied to assess the synergistic and additive action of nine essential oils: caraway, fennel, peppermint, geranium, basil, clove, thyme, clary sage, and lavender, respectively, in combination with gentamicin.

Results:

Our results indicated that peppermint oil combined with gentamicin showed synergistic activity against both control, ESBL-producing and NDM-1-producing isolates. Caraway essential oil demonstrated synergy with gentamicin toward ESBL-producing and additionally gentamicin-resistant strains. The additive effect was observed for gentamicin combined with thyme, fennel, basil, and clary sage.

Conclusions:

Because of their synergistic activity with gentamicin, peppermint, and caraway oils in particular, can be considered as an alternative or an addition for the control of infections with limited therapeutic options due to multidrug resistance.

Introduction

Klebsiella pneumoniae is one of the most important causes of nosocomial infections. It is responsible for a significant proportion of hospital-acquired infections, including septicemias, urinary tract infections, pneumonias, and soft tissue infections primarily affecting immunocompromised patients.¹ The hands of hospital personnel and the gastrointestinal tract of hospitalized patients are the most important reservoirs for the transmission. Outbreaks of multidrug-resistant K. pneumoniae strains have created a real threat for healthcare systems in recent years.² Among K. pneumoniae strains, the most dangerous antibiotic resistance mechanism is represented by carbapenemases production. Infections caused by such strains are associated with high morbidity and mortality, ranging between 20% and 72%.^3,4 It has been proven that resistant strain infections result in higher mortality rate compared with strains susceptible to carbapenems.⁵ There are only a few antibiotics to which those pathogenic microorganisms remain susceptible: polymyxin, tigecycline, or aminoglycosides.⁶ Simultaneously these antibiotics, especially aminoglycosides, remain accepted as a first-choice empirical treatment in certain clinical situations; for example, suspected vascular access infection in hemodialyzed patients. This is also valid for multidrug-resistant Klebsiella infections, if minimum inhibitory concentration for gentamicin is recognized below ≤2 μg/mL.⁷ Still, because of the limited therapeutic options, carbapenemase-producing K. pneumoniae strains will be the major threat to public health in the foreseeable future.

In the era of an increasing bacterial drug resistance, and thus, difficulties in the treatment of infections caused by resistant strains, there has been great interest in plant-derived substances, such as caraway, fennel, peppermint, geranium, basil, clove, thyme, clary sage, and lavender essential oils. According to available data, the essential oils mentioned above have been shown presenting antibacterial activities.^8–11 The search of alternative substances to enhance the efficiency of drug-resistant bacteria elimination becomes a great interest of the scientific community. Therefore, development of combinations containing conventional drug and plant secondary metabolites may appear a novel approach in controlling resistant pathogens, such as carbapenemase-resistant K. pneumoniae (e.g., New Delhi metallo-β-lactamase [NDM] producing) and extended-spectrum β-lactamases (ESBLs)-producing K. pneumoniae. Such studies are considered interesting because it is assumed that multidrug-resistant bacteria will be challenged with conventional antibiotics combined with natural agents.

The aim of the study

The aim of this study was to investigate possible synergistic actions between several selected commercially essential oils and gentamicin against ESBL-producing and NDM-1-producing K. pneumoniae isolates.

Materials and Methods

Bacterial strains and growth condition

The study included four ESBL-producing K. pneumoniae strains belonging to the Department of Microbiology, Immunology, and Laboratory Medicine, Pomeranian Medical University in Szczecin, Poland collection. The strains were identified with GN VITEK^® 2 Compact (bioMérieux, Poland). ESBL production was confirmed by double-disk synergy test.¹² Two strains were positive for bla_NDM-1 gene. K. pneumoniae ATCC^® BAA-1705™ (K. pneumoniae carbapenemase [KPC]-positive) strain was used as a control. Bacteria were cultured in Columbia agar with 5% sheep blood (bioMérieux, Poland) and incubated at 37°C in aerobic atmosphere for 18 hours.

Chemical analysis of the essential oils

Commercial essential oils from Pollena-Aroma (Poland): caraway (Carum carvi L.), fennel (Foeniculum vulgare Mill.), peppermint (Mentha × piperita L.), geranium (Pelargonium graveolens L'Hér.), basil (Ocimum basilicum L.), clove (Syzygium aromaticum (L.) Merrill & Perry), thyme (Thymus vulgaris L.), clary sage (Salvia sclarea L.), and lavender (Lavandula angustifolia Mill.) essential oils were used in this study. Caraway and fennel essential oils were analyzed by gas chromatography–mass spectrometry (GC-MS) at the Faculty of Chemical Technology and Engineering, West Pomeranian University of Technology in Szczecin, Poland, whereas peppermint, geranium, basil, clove, thyme, clary sage, and lavender essential oils were analyzed by gas chromatography with flame ionization detection (GC-FID) in the Institute of General Food Chemistry, Lodz University of Technology, Poland.^11,13,14

Determination of minimal inhibitory concentration and minimal bactericidal concentration of essential oils

The minimal inhibitory concentration (MIC) (the lowest concentration of an antimicrobial that will inhibit the visible growth of a microorganism after overnight incubation) values of the essential oils against K. pneumoniae strains were determined by broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI), with the following modification: a final concentration of 1.0% (v/v) Tween^® 80 (Sigma-Aldrich, Germany) (filter sterilized) was incorporated into the medium to enhance oil solubility.^15,16 Two-fold dilutions (500–3.91 μL/mL) were performed; each well containing 50 μL of the tested essential oil and 50 μL of bacterial suspension at a final concentration of 10⁶ CFU/mL. Bacterial suspensions were prepared from 18-hour cultures using saline. All tests were performed in duplicate. The MIC was estimated after 18 hours of incubation at 37°C in Mueller-Hinton broth (MHB) (Sigma-Aldrich, Germany) using resazurin (Sigma-Aldrich, Germany).¹³ MIC was determined on the basis of the blue color appearance in the first tested well after 3 hours of incubation with resazurin. The color change from blue to pink after 3 hours of incubation with resazurin at 37°C indicated the presence of bacteria. To exclude an inhibitory effect of 1.0% Tween^® 80 on the K. pneumoniae growth, the control assays with MHB and MHB supplemented with 1.0% Tween 80 were performed. Using the known densities of essential oils, the final result was expressed in mg/mL.

The minimal bactericidal concentration (MBC) (the lowest concentration of antimicrobial that will prevent the growth of an organism after subculture on to antibiotic-free media) was determined by transferring all the cultures in higher-than-MIC concentrations on Mueller-Hinton agar (bioMérieux, Poland) and incubating them at 37°C for 18 hours.¹⁵ After incubation time, the MBC was identified as the concentration on which no colony growth was observed.

Determination of MIC and MBC of gentamicin

The MIC values of gentamicin (Sigma-Aldrich, Germany) against K. pneumoniae strains were determined by broth microdilution method according to CLSI.¹⁶ Stock solution of gentamicin was prepared in MHB in a concentration ranging between 3125 and 0.02 mg/L. The MIC and MBC of gentamicin was determined as described above. MIC results were classified as susceptible, intermediate, or resistant based on the EUCAST breakpoints (MIC ≤2 mg/L, susceptible; MIC >2 and MIC ≤4 mg/L, intermediate; MIC >4 mg/L, resistant).⁷

Checkerboard method

The checkerboard method was performed according to Yap et al.¹⁷ with the following modifications: the final concentration of bacterial suspension was 10⁶ CFU/mL and resazurin was added to each plate well after 18 hours of incubation. In the cases of gentamicin susceptible and intermediate strains, the final concentration of gentamicin was recognized as 6.1–0.02 mg/L MHB. In the case of gentamicin-resistant strains, the final concentration of gentamicin was recognized as 3125–0.02 mg/L MHB. Each well contained the following components: 25 μL of the appropriate concentration of essential oil, 25 μL of the appropriate gentamicin concentration, and 50 μL of bacterial suspension containing final concentration of 10⁶ CFU/mL in each well. The plates were incubated at 37°C for 18 hours. All assessments were performed in duplicates. Using the known densities of essential oils, the final result was expressed in mg/mL. The MIC of both gentamicin and essential oils combined or without combination were determined as described above. The combined effects of the antibiotic and essential oils were calculated and expressed in terms of a fractional inhibitory concentration index (FICI) using the following formula: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} { \rm FIC } = { \frac { \rm MIC \ of \ essential \ oil \ or \ antibiotic \ in \ combination } { \rm MIC \ of \ essential \ oil \ or \ antibiotic \ alone } } \end{align*} \end{document} \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} {\rm FICI} =\hbox{FIC of essential oil + FIC of antibiotic} \end{align*} \end{document}

Results were interpreted as synergy (FICI <0.5), addition (0.5 ≤ FICI ≤1.0), indifference (1.1 < FICI ≤4.0), or antagonism (FICI >4.0).¹⁸

Results

Constituents of essential oils

The GC-MS-based study method revealed carvone (52.3%) and limonene (46.8%) as the main components of caraway essential oil. In turn, the predominant constituents of fennel essential oil were trans-anethole (77.9%), fenchone (12.8%), and α-pinene (3.8%). Among the constituents identified in peppermint essential oil, menthol (42.5%), menthone (24.8%), menthyl acetate (4.4%), and isomenthone (3.7%) appeared predominant. The main components of geranium essential oil were citronellol (26.7%), geraniol (13.4%), nerol (8.7%), citronellyl formate (7.1%), isomenthone (6.3%), linalool (5.2%), and 10-epi-γ-eudesmol (4.4%), whereas the major components of basil essential oil were estragole, 1,8-cineole, and trans-α-bergamotene with 86.4%, 4.9%, and 3.0% of relative content, respectively. Clove essential oil consisted primarily of eugenol (85.3%) and β-caryophyllene (10.6%). In the thyme essential oil, thymol (38.1%), p-cymene (29.1%), γ-terpinene (5.2%), and linalool (3.1%) were mainly found. Clary sage oil contained linalyl acetate (57.9%), linalool (12.4%), α-pinene (4.5%), α-terpineol (3.5%), sabinene (3.3%), and β-pinene (3.0%). The main constituents of lavender essential oil were linalool (34.1%), linalyl acetate (33.3%), (Z)-β-ocimene (3.2%), and lavandulil acetate (3.2%). The detailed data of essential oils' composition are shown in Table 1.

Table 1.

The Chemical Composition of Essential Oils Investigated in the Study

	Relative content (%) of essential oils
Compound	Caraway	Fennel	Peppermint	Geranium	Basil	Clove	Thyme	Clary sage	Lavender
Limonene	46.8	2.1	2.2	—	—	—	—	—	—
α-Pinene	—	3.8	—	—	—	—	1.9	4.5	—
Myrcene	—	—	—	—	—	—	1.1	1.5	2.4
β-Caryophyllene	—	—	1.5	1.5	—	10.6	3.1	—	2.7
β-Pinene	—	—	1.3	—	—	—	—	3.0	—
Camphene	—	—	—	—	—	—	1.2	—	—
Linalool	—	—	—	5.2	1.2	—	3.7	12.4	34.1
α-Humulene	—	—	—	—	—	2.0	—	—	—
γ-Terpinene	—	—	—	—	—	—	5.2	—	—
α-Terpineol	—	—	—	—	—	—	—	3.5	1.8
p-Cymene	—	—	—	—	—	—	29.1	—	—
(Z)-β-Ocimene	—	—	—	—	—	—	—	—	3.2
1,8-Cineole	—	—	1.8	—	4.9	—	2.1	—	2.5
β-Bourbonene	—	—	—	1.1	—	—	—	—	—
Terpinen-4-ol	—	—	—	—	—	—	1.3	—	2.5
Sabinene	—	—	—	—	—	—	—	3.3	—
Borneol	—	—	—	—	—	—	1.9	—	1.4
Camphor	—	—	—	—	—	—	—	—	1.2
Estragole	—	2.3	—	—	86.4	—	—	—	—
(E)-β-Ocimene	—	—	—	—	—	—	—	—	2.7
Menthone	—	—	24.8	1.6	—	—	—	—	—
Isomenthone	—	—	3.7	6.3	—	—	—	—	—
Geranyl acetate	—	—	—	—	—	—	—	1.6	1.3
Carvone	52.3	—	—	—	—	—	—	—	—
Fenchone	—	12.8	—	—	—	—	—	—	—
trans-α-Bergamotene	—	—	—	—	3.0	—	—	—	—
Menthol	—	—	42.5	—	—	—	—	—	—
Nerol	—	—	—	8.7	—	—	—	—	—
trans-Anethole	—	77.9	—	—	—	—	—	—	—
Menthofuran	—	—	2.8	—	—	—	—	—	—
Neomenthol	—	—	2.9	—	—	—	—	—	—
Pulegone	—	—	1.4	—	—	—	—	—	—
Menthyl acetate	—	—	4.4	—	—	—	—	—	—
cis-Rose oxide	—	—	—	1.4	—	—	—	—	—
Citronellol	—	—	—	26.7	—	—	—	—	—
Citronellyl formate	—	—	—	7.1	—	—	—	—	-
10-epi-γ-Eudesmol	—	—	—	4.4	—	—	—	—	—
Geraniol	—	—	—	13.4	—	—	—	—	—
Geranial	—	—	—	1.1	—	—	—	—	—
Geranyl formate	—	—	—	2.5	—	—	—	—	—
Geranyl propionate	—	—	—	1.0	—	—	—	—	—
Geranyl butyrate	—	—	—	1.4	—	—	—	—	—
Geranyl tiglate	—	—	—	1.0	—	—	—	—	—
Eugenol	—	—	—	—	—	85.3	—	—	—
Oct-1-en-3-ol	—	—	—	—	—	—	1.0	—	—
Thymol	—	—	—	—	—	—	38.1	—	—
Thymol methyl ether	—	—	—	—	—	—	1.3	—	—
Carvacrol	—	—	—	—	—	—	2.3	—	—
Carvacrol methyl ether	—	—	—	—	—	—	1.0	—	—
Linalyl acetate	—	—	—	—	—	—	—	57.9	33.3
Octan-3-one	—	—	—	—	—	—	—	—	1.3
Lavandulil acetate	—	—	—	—	—	—	—	—	3.2
Lavandulol	—	—	—	—	—	—	—	—	1.1

Constituents present in amount >1% were included.

The antimicrobial effects of gentamicin and essential oils

The results showed that two ESBL-producing and NDM-1-producing isolates were intermediately susceptible to gentamicin with MIC = 3.1 ± 0.0 mg/L. The other two strains producing only ESBLs expressed higher resistance to gentamicin ranging from 293.0 ± 138.1 to 585.9 ± 276.2 mg/L. Moreover, it has been shown that K. pneumoniae ATCC^® BAA-1705™ strain was susceptible to gentamicin with MIC = 1.5 ± 0.0 mg/L.

Both control and clinical isolates were sensitive to tested essential oils. The highest inhibiting activity against clinical isolates was observed for thyme essential oil (MIC: 3.6 ± 0.0–5.4 ± 0.0 mg/mL). In contrast, the lowest antibacterial activity of clary sage essential oil (MIC: 55.6 ± 0.0–222.5 ± 0.0 mg/mL) was observed. Similar results were obtained for the control strain with MIC: 5.4 ± 2.5 mg/mL for thyme and 225.5 ± 0.0 mg/mL for clary sage essential oils. The results of the MICs and MBCs of gentamicin and essential oils against K. pneumoniae strains are summarized in Table 2.

Table 2.

Fractional Inhibitory Concentration and Fractional Inhibitory Concentration Indices of Gentamicin–Essential Oil Pairs Against Klebsiella pneumoniae-Tested Strains

Strains	Phenotypic resistant	Gentamicin–essential oil	MIC_O	MBC	MIC_C	FIC	FICI	Type of interaction
K. pneumoniae ATCC^® BAA-1705™	KPC	Gentamicin–caraway
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	3.1	2.0	2.3	Indifference
		Caraway (mg/mL)	90.4 ± 42.6	120.5 ± 0.0	30.1	0.3	2.3	Indifference
		Gentamicin–fennel
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	1.5	1.0	1.5	Indifference
		Fennel (mg/mL)	120.5 ± 0.0	120.5 ± 0.0	60.3	0.5	1.5	Indifference
		Gentamicin–peppermint
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	0.2	0.1	0.2	Synergy
		Peppermint (mg/mL)	169.0 ± 79.7	>450.5	14.1	0.1	0.2	Synergy
		Gentamicin–geranium
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	3.1	2.0	2.0	Indifference
		Geranium (mg/mL)	166.9 ± 78.6	>445.0	7.0	0.0	2.0	Indifference
		Gentamicin–basil
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	3.1	2.0	2.5	Indifference
		Basil (mg/mL)	29.5 ± 0.0	177.2 ± 83.6	14.8	0.5	2.5	Indifference
		Gentamicin–clove
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	3.1	2.0	3.0	Indifference
		Clove (mg/mL)	8.0 ± 0.0	8.0 ± 0.0	8.0	1.0	3.0	Indifference
		Gentamicin–thyme
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	0.1	0.0	0.7	Addition
		Thyme (mg/mL)	5.4 ± 2.5	7.1 ± 0.0	3.6	0.7	0.7	Addition
		Gentamicin–clary sage
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	3.1	2.0	3.0	Indifference
		Clary sage (mg/mL)	222.5 ± 0.0	>445.0	222.5	1.0	3.0	Indifference
		Gentamicin–lavender
		Gentamicin (mg/L)	1.5 ± 0.0	6.1 ± 0.0	3.1	2.0	6.0	Antagonism
		Lavender (mg/mL)	13.7 ± 0.0	219.5 ± 0.0	54.9	4.0	6.0	Antagonism
5404	ESBL NDM-1	Gentamicin–caraway
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	3.1	1.0	2.0	Indifference
		Caraway (mg/mL)	60.3 ± 0.0	180.8 ± 85.2	60.3	1.0	2.0	Indifference
		Gentamicin–fennel
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	6.1	2.0	3.0	Indifference
		Fennel (mg/mL)	60.3 ± 0.0	90.4 ± 42.6	60.3	1.0	3.0	Indifference
		Gentamicin–peppermint
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	0.2	0.1	0.1	Synergy
		Peppermint (mg/mL)	169.0 ± 79.7	>450.5	7.0	0.0	0.1	Synergy
		Gentamicin–geranium
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	3.1	1.0	1.5	Indifference
		Geranium (mg/mL)	111.3 ± 0.0	445.0 ± 0.0	55.6	0.5	1.5	Indifference
		Gentamicin–basil
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	3.1	1.0	2.0	Indifference
		Basil (mg/mL)	29.5 ± 0.0	354.4 ± 167.0	29.5	1.0	2.0	Indifference
		Gentamicin–clove
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	0.8	0.3	1.6	Indifference
		Clove (mg/mL)	6.0 ± 2.8	16.1 ± 0.0	8.0	1.3	1.6	Indifference
		Gentamicin–thyme
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	0.4	0.1	1.2	Indifference
		Thyme (mg/mL)	3.6 ± 0.0	5.4 ± 2.5	4.0	1.1	1.2	Indifference
		Gentamicin–clary sage
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	3.1	1.0	1.7	Indifference
		Clary sage (mg/mL)	166.9 ± 78.6	>445.0	111.3	0.7	1.7	Indifference
		Gentamicin–lavender
		Gentamicin (mg/L)	3.1 ± 0.0	4.6 ± 2.2	6.1	2.0	6.0	Antagonism
		Lavender (mg/mL)	13.7 ± 0.0	82.4 ± 38.8	54.9	4.0	6.0	Antagonism
10245	ESBL NDM-1	Gentamicin–caraway
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	6.1	2.0	2.7	Indifference
		Caraway (mg/mL)	90.4 ± 42.6	241.0 ± 0.0	60.3	0.7	2.7	Indifference
		Gentamicin–fennel
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	6.1	2.0	2.5	Indifference
		Fennel (mg/mL)	120.5 ± 0.0	120.5 ± 0.0	60.3	0.5	2.5	Indifference
		Gentamicin–peppermint
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	0.2	0.1	0.2	Synergy
		Peppermint (mg/mL)	169.0 ± 79.7	>450.5	14.1	0.1	0.2	Synergy
		Gentamicin–geranium
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	3.1	1.0	1.2	Indifference
		Geranium (mg/mL)	111.3 ± 0.0	445.0 ± 0.0	27.8	0.2	1.2	Indifference
		Gentamicin–basil
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	6.1	2.0	3.3	Indifference
		Basil (mg/mL)	22.2 ± 10.4	118.1 ± 0.0	29.5	1.3	3.3	Indifference
		Gentamicin–clove
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	1.5	0.5	2.5	Indifference
		Clove (mg/mL)	8.0 ± 0.0	8.0 ± 0.0	16.1	2.0	2.5	Indifference
		Gentamicin–thyme
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	0.1	0.0	0.7	Addition
		Thyme (mg/mL)	5.4 ± 2.5	7.1 ± 0.0	3.6	0.7	0.7	Addition
		Gentamicin–clary sage
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	1.5	0.5	1.5	Indifference
		Clary sage (mg/mL)	222.5 ± 0.0	>445.0	222.5	1.0	1.5	Indifference
		Gentamicin–lavender
		Gentamicin (mg/L)	3.1 ± 0.0	6.1 ± 0.0	6.1	2.0	6.0	Antagonism
		Lavender (mg/mL)	13.7 ± 0.0	41.2 ± 19.4	54.9	4.0	6.0	Antagonism
35214	ESBL	Gentamicin–caraway
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	6.1	0.0	0.2	Synergy
		Caraway (mg/mL)	45.2 ± 21.4	120.5 ± 0.0	7.5	0.2	0.2	Synergy
		Gentamicin–fennel
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	48.8	0.1	0.8	Addition
		Fennel (mg/mL)	45.2 ± 21.4	60.3 ± 0.0	30.1	0.7	0.8	Addition
		Gentamicin–peppermint
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	1562.5	2.7	3.7	Indifference
		Peppermint (mg/mL)	3.5 ± 0.0	5.3 ± 2.5	3.5	1.0	3.7	Indifference
		Gentamicin–geranium
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	1562.5	2.7	5.3	Antagonism
		Geranium (mg/mL)	5.3 ± 2.5	41.7 ± 19.7	13.9	2.6	5.3	Antagonism
		Gentamicin–basil
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	195.3	0.3	0.6	Addition
		Basil (mg/mL)	14.8 ± 0.0	177.2 ± 83.6	3.7	0.3	0.6	Addition
		Gentamicin–clove
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	1562.5	2.7	2.8	Indifference
		Clove (mg/mL)	4.0 ± 0.0	6.0 ± 2.8	0.5	0.1	2.8	Indifference
		Gentamicin–thyme
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	1562.5	2.7	3.2	Indifference
		Thyme (mg/mL)	3.6 ± 0.0	5.4 ± 2.5	1.8	0.5	3.2	Indifference
		Gentamicin–clary sage
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	97.7	0.2	0.9	Addition
		Clary sage (mg/mL)	166.9 ± 78.6	445.0 ± 0.0	111.3	0.7	0.9	Addition
		Gentamicin–lavender
		Gentamicin (mg/L)	585.9 ± 276.2	9375.0 ± 4419.4	195.3	0.3	2.3	Indifference
		Lavender (mg/mL)	6.9 ± 0.0	13.7 ± 0.0	13.7	2.0	2.3	Indifference
2725	ESBL	Gentamicin–caraway
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	1.5	0.0	0.3	Synergy
		Caraway (mg/mL)	22.6 ± 35.1	120.5 ± 0.0	7.5	0.3	0.3	Synergy
		Gentamicin–fennel
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	195.3	0.7	1.0	Addition
		Fennel (mg/mL)	45.2 ± 21.4	60.3 ± 0.0	15.1	0.3	1.0	Addition
		Gentamicin–peppermint
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	390.6	1.3	1.6	Indifference
		Peppermint (mg/mL)	3.5 ± 0.0	5.3 ± 2.5	0.9	0.3	1.6	Indifference
		Gentamicin–geranium
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	781.3	1.3	3.9	Indifference
		Geranium (mg/mL)	5.3 ± 2.5	7.0 ± 0.0	13.9	2.6	3.9	Indifference
		Gentamicin–basil
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	195.3	0.7	0.8	Addition
		Basil (mg/mL)	14.8 ± 0.0	177.2 ± 83.6	0.9	0.1	0.8	Addition
		Gentamicin–clove
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	390.6	1.3	1.4	Indifference
		Clove (mg/mL)	4.0 ± 0.0	8.0 ± 0.0	0.5	0.1	1.4	Indifference
		Gentamicin–thyme
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	195.3	0.7	0.8	Addition
		Thyme (mg/mL)	3.6 ± 0.0	5.4 ± 2.5	0.4	0.1	0.8	Addition
		Gentamicin–clary sage
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	390.6	1.3	1.6	Indifference
		Clary sage (mg/mL)	55.6 ± 0.0	111.3 ± 0.0	13.9	0.3	1.6	Indifference
		Gentamicin–lavender
		Gentamicin (mg/L)	293.0 ± 138.1	390.6 ± 0.0	781.3	2.7	2.8	Indifference
		Lavender (mg/mL)	6.9 ± 0.0	13.7 ± 0.0	0.9	0.1	2.8	Indifference

FIC index = FIC of oil + FIC of gentamicin.

FICI <0.5, synergy; 0.5 ≤ FICI ≤1.0, addition; 1.1 < FICI ≤4.0, indifference; FICI >4.0, antagonism.

ESBL, extended-spectrum β-lactamases; FIC, fractional inhibitory concentration; FICI, fractional inhibitory concentration index; KPC, Klebsiella pneumoniae carbapenemase; MBC, minimal bactericidal concentration; MICc, MIC of one sample of the most effective combination; MICo, MIC of one sample alone; NDM-1, New Delhi metallo-β-lactamase-1.

Combination of essential oils and gentamicin

Our studies indicated that peppermint oil showed synergistic activity with gentamicin against control (susceptible to gentamicin), ESBL-producing, and NDM-1-producing isolates (intermediate to gentamicin). Other results were obtained for gentamicin-resistant ESBL-producing strains. For these, synergistic action was demonstrated by caraway essential oil. Following essential oils, thyme, fennel, basil, and clary sage, exhibited additive effect in combination with gentamicin. Lavender essential oil presented antagonistic effect when used with gentamicin against control strain and also against ESBL-producing and NDM-1-producing isolates. Also geranium essential oil exhibited antagonistic effect against one strain of K. pneumoniae resistant to gentamicin. Overall, a number of combinations showed indifferent effect. The results for checkerboard assay against K. pneumoniae tested strains are listed in Table 2.

Discussion

Currently, many scientific centers draw attention to the natural products, such as plant secondary metabolites. Due to the development of modern methodology it has become possible to recognize their mode of action or side effects more precisely. Antimicrobial properties of these have been used in traditional medicine, food preservation, and in cosmetic industry.^19,20 At the same time, it appears that conventional, synthetic drugs do not fully meet therapeutic expectations. This has been clearly revealed that the old and relatively cheap drugs, which have been abandoned due to their toxicity, are now becoming of increasing interest among clinicians as the last chance treatment. This strongly refers to aminoglycosides, previously praised for their synergy with β-lactams, but less commonly used due to their nephrotoxicity. However as mentioned above, in certain groups, such as hemodialyzed patients, these antibiotics are still considered as the first-line empirical treatment of systemic infections, which is able to cover possible multidrug-resistant Gram-negative bacilli strains.

We decided to investigate the potential of synergistic activity of the well-known essential oils in combination with gentamicin, because these oils have been precisely described in the monographs published in the European Pharmacopoeia (EP) and ISO norms, as well as in WHO and Council of Europe documents, which supply clinical users with necessary information. On the other hand, knowing the reasons which prevent clinicians from common use of aminoglycosides as well as the limited but still valid application of these antibiotics in the highly specified group, we found intriguing if any naturally derived substances may possibly enhance their activity, consequently reduce required dosage, and therefore make them more efficient without the risk of adverse events.

Mahboubi and Kazempour²¹ showed synergism of peppermint oil with vancomycin, gentamicin, and amphotericin B against the reference strains of Staphylococcus aureus, Escherichia coli, Candida albicans, and Aspergillus niger. Moreover, the composition of used essential oils was similar to peppermint oil used in this study. Schelz et al.²² reported that peppermint essential oil preferentially kills bacteria carrying resistance F'lac plasmids. Therefore, Yap et al.¹⁷ indicate that peppermint oil could reduce plasmid-conferred meropenem antibiotic resistance in E. coli. This corresponds with our finding of peppermint oil susceptibility of the NDM-1-producing isolates.

Apart from that, relevant publication reports the synergistic activity of carvone–penicillin and thymol–penicillin combinations, respectively, against S. aureus and E. coli.²³ Sieniawska et al.²⁴ showed that limonene—the primary constituent of caraway oil showed synergistic activity against Mycobacterium tuberculosis isolate together with tuberculostatic antibiotics, such as ethambutol, isoniazid, and rifampicin. In our observations, additive effect against tested ESBL-producing strains of K. pneumoniae was drawn for combination of gentamicin with fennel, basil, and clary sage oil. Combinations of clary sage, basil, and rosemary oil were also found beneficial against gentamicin-resistant Enterococcus faecalis and Acinetobacter baumannii isolates.¹¹ Trombetta et al.²⁵ noted that monoterpenes lead to deviation of the lipid fraction of bacteria cell membranes, which results in increasing membrane permeability and, consequently, intracellular homeostasis disruption. Increased cytoplasmic gentamicin concentration enhanced their activity furthermore. Moreover, other research suggests that plant extracts which contained tannins, terpenoids, alkaloids, glycosides, saponins, and flavonoids could inhibit the NDM-1 enzyme activity in vitro, possibly by chelation of zinc ions, which are essential for catalytic activity of this enzyme.^26,27

The present study confirms the potency of essential oils—unique and special natural products of plants to synergistic and additive action with antibiotics. According to our investigations, the ability to synergize with gentamicin was shown by the essential oils with standard reliably confirmed compositions described by EP: peppermint (ISO-856) and caraway (ISO-8896) oils. Other, considered as safe, such as essential oils from Lamiaceae family— thyme (ISO-19817), basil (ISO-11043), clary sage, and from Apiaceae family—fennel oil (ISO-17412) demonstrated the additive effect.²⁸

In summary, our studies indicate that essential oils, such as peppermint, caraway, thyme, basil, clary sage, and fennel, can be considered useful to develop the specific aminoglycoside (gentamicin)-essential oil combination to reduce the resistance of ESBL-producing and NDM-1-producing K. pneumoniae isolates from hospital milieu. Peppermint and caraway oils merit special attention and should be recognized as a subject of further bench-to-bed research. Intravenous application of oil components appears unacceptable. However, gentamicin can also be administered intramuscularly, similar to drugs which contain insoluble (also oil) components. In such context possible intramuscular administration of oil combined with antibiotic seems more promising for the eradication of clinically challenging K. pneumoniae strains. Depending on the clinical situation, the use of essential oil—antibiotic combinations in ointments or active dressings—could also be considered.

Footnotes

Acknowledgments

The authors thank the Pomeranian Medical University in Szczecin for their financial support as well as the Microbiology, Immunology, and Laboratory Medicine Department of this Institute. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Disclosure Statement

No competing financial interests exist.

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