Abstract
Due to the increase in bacterial resistance to antimicrobials (AMBs) commonly used in veterinary and human medicine, the new strategies for controlling zoonoses focus on the study of natural products with demonstrated AMB activity, such as essential oils (EOs). The aim of this study was to evaluate the in vitro effect of the combination of enrofloxacin (ENR), ceftiofur (CEF), and trimethoprim–sulfamethoxazole (SXT) with cinnamon, clove, oregano, and red thyme EOs against multiple drug-resistant strains of Salmonella enterica. The minimum inhibitory concentration (MIC) of each product was determined by microdilution and “Checkerboard” methods and their combined effect was evaluated against 15 strains of S. enterica. The results were interpreted by the calculation of fractional inhibitory concentration (FIC) and their respective indexes (FICI). Significant susceptibility of all strains to the four EOs was observed. The results showed a synergistic effect between EOs and AMBs tested, highlighting the upper percentage of total synergies of the SXT with the four EOs (FICI ≤0.5 in 60% of assays), and the most effective combination being the one of ENR and cinnamon. The MIC of cinnamon was reduced from 1250 to 312.5 μg/mL and the MIC of ENR from 2 to 0.031 μg/mL. There was no antagonism in the tested combinations (AMBs-EOs). Our results support the combined use of EOs and AMBs for the control of multiresistant strains of S. enterica with a reduction of the minimum effective dose of AMBs and their adverse effects.
Highlights
• Antimicrobial (AMB)-essential oil (EO) combination on multiresistant Salmonella was studied.
• AMBs assayed: enrofloxacin (ENR), ceftiofur (CEF) and sulfamethoxazole–trimethoprim.
• EOs tested: cinnamon, clove, oregano, and red thyme.
• Synergism was observed in all AMB-EO combinations. The highest minimum inhibitory concentration reduction was observed for ENR-cinnamon combination.
Introduction
U
Moreover, international committees of experts have indicated the need to search new alternatives for the treatment of infectious diseases (Randall et al., 2016). Among the substances studied with the highest potential are essential oils (EOs), extracted from plants and spices, authorized as animal food supplements, for which bactericidal, anti-inflammatory, immunostimulating, and anticancer properties have been previously described (Becerril et al., 2012; Yap et al., 2014). Among these, the EOs of cinnamon, clove, oregano, and red thyme have shown remarkable activity against Gram-positive (Huerta et al., 2016) and Gram-negative bacteria of interest in public and animal health (i.e., Listeria spp., Staphylococcus aureus, Salmonella enterica and Escherichia coli). The main feature of EOs is their complex chemical composition and the synergistic interaction of many of their active ingredients. The action of EOs is developed by various mechanisms that affect bacterial survival, which decrease the possibility of resistant strain selection (Nazzaro et al., 2013).
Langeveld et al. (2014) suggested a possible change of the bacterial resistance mechanism for AMBs, consisting in the formation of permeability barriers and/or efflux pumps, because of the rupture of the cell membrane by inhibition of Adenosine Triphosphatase (ATPase) by cinnamaldehyde. Another plausible explanation is that after entering the bacterial cell, the plant molecules inhibited the efflux pumps responsible for reduced antibiotic concentration within the cell (Kollanoor-Johny et al., 2010). However, a dose-dependent cytotoxic effect on the intestinal cells has been described (Dušan et al., 2006). Studies on the combined effect of EOs and AMBs highlight this combination as a possible solution to the problem of AMB resistance (Liu et al., 2014).
The great advantage of using EOs is that they can be easily added to feed as dietary supplementation, by means of encapsulated blends (Walia et al., 2017) or as biopreservatives, with the aim of increasing the overall quality of feed (Solórzano-Santos and Miranda-Novales, 2012).
The objective of this research was to evaluate the synergistic effect of the combination of the ENR, CEF, and SXT with the EOs of cinnamon, clove, oregano, and red thyme against S. enterica MDR strains from animal source.
Materials and Methods
Antimicrobials
The AMBs studied were ENR (High Performance Liquid Chromatography [HPLC] 98% purity), CEF hydrochloride (HPLC >95% purity), and SXT (HPLC 99.7% purity), all of Sigma Aldrich Co. Each AMB was dissolved following the parameters set described in Clinical and Laboratory Standards Institute until obtaining a stock solution of 1024 μg/mL (CLSI, 2015).
Essential oils
The scientific name, main active compounds, and registry number of the EOs tested were as follows: cinnamon (Cinnamomum zeylanicum) (Cinnamaldehyde) AR007AE, clove (Eugenia caryophyllata) (Eugenol, Eugenyl acetate) AR014AE10, oregano (Origanum vulgare) (Thymol, Carvacrol) AR071AE10, and red thyme (Thymus zygis) (Thymol, p-cymene, g-terpinene, and linalool) AR189AE05. They were obtained from the same factory, with purity higher than 98% (Aromium S.L. Barcelona, Spain). The chemical composition of the oils was determined and provided by the manufacturer.
Bacterial strains
For this study, 15 clinical strains of S. enterica, previously characterized as MDR, were selected from the Animal Health Department Culture Type Collection (Veterinary Faculty, University of Cordoba, Spain) and Central Veterinary Laboratory (Algete, Madrid, Spain). Each AMB was tested against five strains. The selected strains were obtained from different animal species (chicken, turkey, partridge, horse, and pig) and belonged to the following serotypes: Typhimurium, Enteritidis, London, and Hadar.
The reference strains E. coli ATCC 25922 and Salmonella Typhimurium ATCC 14028 of American Type Culture Collection (ATCC, Manassas) were included as quality controls.
Determination of individual minimum inhibitory concentrations of EOs and AMBs against every studied strain
Following the broth microdilution method (CLSI, 2015), double serial dilutions of the pure EOs and stock solution of AMBs were prepared in Müller-Hinton (MH) broth: final concentration range for EOs 10,000–78.125 μg/mL and final concentration range for AMBs 32–0.0625 μg/mL. Each dilution of EOs and each dilution of AMBs were challenged with an equal volume of bacterial inoculum up to a final concentration of 5 × 105 CFU/mL, using a-96 well plate. Tests were conducted in triplicate including positive growth controls (MH broth without EO, but with bacterial inoculum) and negative controls (MH broth without EO and without inoculum). After incubation at 37°C for 16–20 h, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of EO or AMB capable of inhibiting the visible growth of bacteria in the wells.
Assessment of the combined effect of EOs and AMBs
This assay was performed following the Checkerboard technique (Si et al., 2008). Seven serial twofold dilutions of each AMB and 11 serial twofold dilutions of each EO were prepared as in the MIC tests, considering the MICI results of every single strain.
A volume of 50 μL of each AMB- and EO-tested solutions was mixed and inoculated with 100 μL of bacterial suspension containing final concentration of 5 × 105 CFU/mL. The microtiter plates were incubated at 37°C for 16–20 h. All the assays included positive and negative growth controls for each strain. The MICI of both AMBs and EOs and the combined MIC (MICC) were determined as described above.
Calculation of fractional inhibitory concentration and fractional inhibitory concentration index
The effect of the combination of each AMB and EO was established and expressed by calculation of fractional inhibitory concentration (FIC) and fractional inhibitory concentration index (FICI) according to the following formulas (Knezevic et al., 2016):
The interpretation of the results was performed according to the criteria of Basri et al. (2014): total synergistic effect is defined as FICI of ≤0.5; partial synergism as >0.5 < 1; additivity as 1; indifference as >1 ≤4; and antagonism as FICI >4.
Results
Individual MICI
Tables 1 –3 show the MICI obtained for each EO and each AMB against the five selected strains. The MICI of ENR, CEF, and SXT was 2, 4, and 8–16 μg/mL, respectively. The MICI of oregano and red thyme EOs ranged between 312.5 and 625 μg/mL and of cinnamon and clove ranged between 625 and 1250 μg/mL.
Total synergistic effect: FICI ≤0.5; partial synergism: FICI >0.5 < 1; additivity: FICI 1; indifference: FICI >1 ≤4; antagonism: FICI >4. FICI values are signified in bold.
ENR, enrofloxacin; FICI, fractional inhibitory concentration index; MIC, minimum inhibitory concentration; MICI, individual minimum inhibitory concentration (μg/mL); MICC, combined MIC (μg/mL); SXT, trimethoprim–sulfamethoxazole.
Total synergistic effect: FICI ≤0.5; partial synergism: FICI >0.5 < 1; additivity: FICI 1; indifference: FICI >1 ≤4; antagonism: FICI >4. FICI values are signified in bold.
CEF, ceftiofur; FICI, fractional inhibitory concentration index; MIC, minimum inhibitory concentration; MICI, individual minimum inhibitory concentration (μg/mL); MICC, combined MIC (μg/mL).
Total synergistic effect: FICI ≤0.5; partial synergism: FICI >0.5 < 1; additivity: FICI 1; indifference: FICI >1 ≤4; antagonism: FICI >4. FICI values are signified in bold.
ENR, ENRFICI, fractional inhibitory concentration index; MIC, minimum inhibitory concentration; MICI, individual minimum inhibitory concentration (μg/mL); MICC, combined MIC (μg/mL).
Combined effect of EOs with each AMB
The results obtained showed synergistic effects of all AMBs with, at least, one of the tested EOs (Tables 1 –3).
The combination of SXT with cinnamon showed a total synergistic effect (FICI <0.5) on three of the five tested strains. The combination of SXT with clove, on four of the five tested strains, and the combination of SXT with oregano gave the same result. Nevertheless, the combination of SXT with red thyme led to a partial synergism (in three of the five tested strains). In most cases, MICI of SXT and each EO resulted in a lower MICC when combining the two. The cinnamon enhanced the action of SXT at a lower dose (0.06 μg/mL) compared to SXT used alone (4 μg/mL) against Salmonella Typhimurium (Table 1).
In case of CEF, in one of the five tested strains, its MICI was reduced from 8 to 0.12 μg/mL when combined with cinnamon, and from 8 to 0.24 μg/mL when combined with red thyme. CEF showed synergism with each tested EO, resulting in a total synergistic effect in two of the five tested strains when combining CEF with cinnamon and also when combining CEF with clove and with red thyme. The combination of CEF with oregano indicated this total synergistic effect only in one of the five tested strains (Table 2).
Finally, ENR showed a total synergistic effect with cinnamon on three of the five tested strains and partial synergism with clove (on one of the five tested strains) and with oregano (on three of the five tested strains). It should be noted that in five of the five strains, the ENR-cinnamon combination resulted in a significant reduction of ENR MICI (from 2 to 0.03125 μg/mL). The combination of ENR with red thyme had an additive or indifferent effect in all tested strains (FICI 1.0–1.015) (Table 3).
Discussion
The possibility of combining natural products with demonstrated AMB properties to reduce selection of resistant strains has focused on the interest of sensitivity studies in recent years. Increased sensitivity of MDR Salmonella strains to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline when coexposed to subinhibitory doses of cinnamaldehyde, thymol, or carvacrol showed the first evidence of their synergistic effect (Kollanoor-Johny et al., 2010). However, most studies have been developed with strains from human origin and AMBs of interest in public health. The objective of this study was to evaluate in vitro effect of the combination of cinnamon, clove, oregano, and red thyme EOs with the main AMBs used for the control of S. enterica MDR strains from animal origin. This research work is therefore one of the first investigations carried out in animal health. The fact that the strains under study have been isolated from real clinical cases differentiates our work from other studies in which only reference strains are used (Knezevic et al., 2016).
There are studies carried out with various species of thyme and ciprofloxacin that have described a variable effect, depending on the botanical species, from synergy absence to total synergistic effect, probably associated with small differences in thyme active components (Fadli et al., 2012). These same researches found a total synergistic effect (FIC ≤0.5) between thyme and cefixime (cephalosporin III generation). The assays carried out in our study with CEF showed synergistic effect with all EOs against, at least, one of the strains of Salmonella spp., although the greatest potentiation was observed in combination with cinnamon.
EOs with a high percentage of thymol and carvacrol (thyme and oregano) have generally shown a greater AMB potential than EOs with cinnamaldehyde (cinnamon) and eugenol (clove) (Bajpai et al., 2012). However, some authors report differences in the activity of cinnamon depending on the bacterial species, with a potential like the one of thyme and oregano against Salmonella Typhimurium (Gill and Holley, 2004), which could explain the synergistic effect detected in our work with cinnamon.
In addition to an evident enhancement of its antibacterial activity, the combination of EOs and AMBs would reduce the adverse effects of these drugs due to a decrease in administration doses.
In previous studies on in vitro sensitivity, cinnamon required concentrations from 625 to 1250 μg/mL to inhibit the growth of all strains tested, remaining above the cytotoxicity limit (500 μg/mL) (Dušan et al., 2006). The reduction observed when combined with AMBs would therefore allow this EO to be used below this limit.
Conclusions
The results of this research show that cinnamon, clove, oregano, and red thyme EOs are effective in improving the sensitivity of clinical animal origin MDR strains of S. enterica to SXT, CEF and ENR. These EOs could be potentially used as feed supplements to reduce the antibiotic resistance in S. enterica in food animals. Although clinical studies are scarce, the uses of EOs combined with AMBs are promising.
No antagonism was found in the tested EO-AMB combinations, which could help to control resistant bacterial subpopulations without using AMB doses higher than MIC.
This work could be considered a preliminary study to know the synergistic effects between EOs and AMBs. Our results could be the basis of further and deepest studies with more clinical strains, not only in vitro but also in vivo.
Footnotes
Disclosure Statement
No competing financial interests exist.