Reduced Susceptibility of Salmonella Typhimurium Strains to Oregano Essential Oil and Enrofloxacin: An In Vitro Assay

Abstract

Several European animal nutrition companies have incorporated essential oils (EOs) into animal feed as a result of the prohibition of antibiotics to promote animal growth. Previous studies of EOs have highlighted the absence of bacterial resistance for these substances, although most of the published works focus on studying their tolerance to subinhibitory doses. For this study, oregano essential oil (OEO) was chosen for its proven inhibitory and bactericidal activity. This study is an in vitro assay of the possible induction of Salmonella enterica serovar Typhimurium strains with reduced susceptibility to OEO by mutation, seeking to calculate the mutant prevention concentration (MPC) since this is an important measurement for the control Salmonella's resistance to fluoroquinolones such as enrofloxacin (ENR), the treatment of choice for this infection. To establish the MPC, we used a bacterial inoculum ≥10⁹ colony-forming unit (CFU)/mL and examined the bases for points of resistance to ENR and mutations of target genes of the quinolone resistance determining region (QRDR). The three strains of Salmonella Typhimurium used in this study showed an MPC of four times the minimum inhibitory concentration (MIC) for ENR. In all cases, strains with reduced susceptibility to ENR were obtained, although none reached the point of resistance. The QRDR characterization region was in all cases of wild type (wt). Two of the strains tested with OEO grew at a concentration of 1 × MIC, which could be strains with reduced susceptibility, associated with mutation or not. In this case, the MPC was 2 × MIC. Once isolated and identified as Salmonella Typhimurium, the MIC against OEO of all strains obtained in the induction test indicated a possible reduction in susceptibility. However, the result obtained for both strains coincided with MIC of the original strains, rejecting a priori such a reduced susceptibility of Salmonella Typhimurium to OEO.

Introduction

The emergence of a resistant strain means that the use of the antimicrobial at the usual dose fails to inhibit its growth. As a result, the dose is increased promoting the emergence of more and more resistant strains until the antimicrobial becomes no longer effective (Chovanová et al., 2015).

To mitigate the problem of bacterial resistance, numerous investigations have been carried out in recent decades to search for new antimicrobials with added value over existing ones (EC, 2011; Gómez-Laguna et al., 2011). The published results highlight the bactericidal activity of essential oils (EOs) extracted from plants and spices and authorized in animal health as food supplements (Yap et al., 2014). Among them, the oregano essential oil (OEO) have shown a remarkable activity against Salmonella enterica serovar Typhimurium, which is of interest in public and animal health. Of particular interest is its synergistic effect with various antibiotics (Si et al., 2008; Becerril et al., 2012) and its capacity to develop multiple actions, each of which could hinder the emergence of bacterial resistance (Nazzaro et al., 2013; Langeveld et al., 2014; Rodríguez-García et al., 2016). OEO was chosen for its proven antimicrobial potential against S. enterica. Also, the presence of strains of Salmonella Typhimurium with a possible profile of resistance to multiple EOs supports new studies on resistance mechanisms. However, the synergistic effect observed between OEO and enrofloxacin (ENR) could be an effective measure of control against multiresistant strains of S. enterica (Solarte et al., 2017, 2018). Likewise, subchronic toxicity studies with OEO, based on oral administration for 90 d of 50–200 mg/kg, did not reveal mortality or other adverse effects (Llana-Ruiz-Cabello et al., 2017).

However, there are few data on this subject, and some investigations have shown a susceptibility reduction in Gram-negative bacteria and Candida spp. after continued exposure to the tea tree, clove, and geranium EOs (Papadopoulos et al., 2008; Budzyńska et al., 2013). The authors associated this resistance with the activation of nonspecific efflux pumps or a chromosomal mutation. “Single-step” mutations in the target genes of quinolone resistance determining region (QRDR) are the most common resistance mechanism of Salmonella Typhimurium against fluoroquinolones (Randall et al., 2016; Lee et al., 2017). The appearance of a resistant strain supposes that the use of the antimicrobial at the usual dose, or lower, fails to inhibit its growth causing a positive selective pressure on the resistant bacterial subpopulation that will lead to therapeutic failure (Marchetti et al., 2011; Langeveld et al., 2014; Chovanová et al., 2015).

Statistically, genetic mutations usually occur every 10^9–10¹⁰ divisions, so the resistant mutant strains constitute a very small percentage within the bacterial population. Nevertheless, these strains can grow at the usual doses of treatment established for a standard inoculum (5 × 10⁵ colony-forming unit [CFU]/mL) (Dahiya et al., 2014; Abraham et al., 2015). Several authors support the need to establish optimal dosage intervals considering the mutant prevention concentration (MPC), understood as the minimum concentration of antimicrobial capable of inhibiting the growth of both susceptible strains and resistant strains by first-pass mutations, which would be present in infections with high bacterial density (Balaje et al., 2013; Nordqvist et al., 2016). This concentration could exceed safety limits for the animal, being based purely on pharmacokinetic and pharmacodynamic parameters (Pasquali and Manfreda, 2007). The range of concentrations between minimum inhibitory concentration (MIC) and MPC, which would inhibit the growth of sensitive subpopulations but would favor the growth of resistant mutants, is known as the mutant selection window (MSW) (Drlica, 2003).

The objective of this research was to make a first assessment on the possible induction of strains of Salmonella Typhimurium under OEO selection pressure and to compare MPC values with those for ENR.

Material and Methods

Bacterial strains

For this study, Salmonella Typhimurium ATCC 14028 and two porcine clinical strains of Salmonella Typhimurium (54UCO001 and 57UCO003), sensitive to ENR and to OEO (Table 1), were selected from the Animal Health Department Culture Type Collection (Veterinary Faculty, University of Cordoba-Spain). All the strains were stored at −20°C and subjected to viability tests (growth curve) before their use in induction test. Escherichia coli ATCC 25922 (American Type Culture Collection, Manassas, VA) was used as quality control strain.

Table 1.

Tested Strains of Salmonella Typhimurium, Phage Type and Resistance Pattern

Strain No.	Origin	Phage type	Resistance pattern	MIC (μg/mL)
Strain No.	Origin	Phage type	Resistance pattern	ENR	OEO
ATCC14028	Reference strain	NT	Neither	0.0625	312.5
54UCO001	Porcine	PNR	SPT CST	0.0625	312.5
57UCO003	Porcine	104b	AMP AMX SPT SU C TE CST NIT	0.0625	312.5

AMP, ampicillin; AMX, amoxicillin; C, chloramphenicol; CST, colistin; ENR, enrofloxacin; MIC, minimum inhibitory concentration; NIT, nitrofurantoin; NT, not typable; OEO, oregano essential oil; PNR, pattern not recognized; SPT, streptomycin; SU, sulfonamide; TE, tetracycline.

Tested antimicrobial agents

A commercial OEO (Origanum vulgare), Class: Magnoliopsida, Order: Lamiales, Family: Lamiaceae, Genus: Origanum L. with purity higher than 98%, was obtained (Aromium S.L. Barcelona, Spain) (Registry number: 130006133). The chemical composition was determined and provided by the manufacturer: carvacrol (63.01%), thymol (10.56%), and γ-terpinene (8.11%).

ENR, ENR-HPLC (98% purity) (Sigma–Aldrich Co., MO), was used as quality control, following the parameters set described in the Clinical and Laboratory Standards Institute (CLSI, 2015).

Determination of MIC

To design the induction test, MICs of original strains were determined by the method of agar dilution (CLSI, 2015), using dilutions of OEO from 1250 to 78.125 μg/mL, and those of ENR from 0.5 to 0.03 μg/mL.

Determination of MPC

Each strain was grown in 50 mL of Müeller–Hinton (MH) broth at 37°C for 24 h to reach a concentration ≥10⁹ CFU/mL (Alemany, 2009). For each tested strain, 200 μL of the concentrated cellular suspension was plated on three Müeller–Hinton Agar (MHA) plates (Oxoid-Ltd.) supplemented with different concentrations of either OEO (1 × to 16 × MIC) or ENR (1 × to 64 × MIC). After incubating the plates at 37°C for 2 d, colonies were counted, and the plates were incubated again for additional 3 d. The MPC was recorded as the lowest antimicrobial concentration preventing the emergence of any mutant after 2 or 5 d of incubation (Pasquali and Manfreda, 2007). Each experiment was carried out in triplicate, the colonies with growth at sub-MPC concentrations were selected for tests performed with the relevant strain. In each case, up to two colonies per plate were selected for induction testing. The induction tests included two positive growth controls (two MHA plates without OEO and two MHA plates without ENR supplementation) to verify growth of strains in MHA. In addition, two quality controls were included (two plates supplemented with OEO and two plates supplemented with ENR in sub-MIC concentrations), which served as control of the behavior of the strain against OEO and ENR.

Some strains were randomly selected from plates supplemented with a product concentration equal to or higher than MIC and lower than the MPC (within the MSW). These strains were cultured on OEO- and ENR-free agar plates and submitted to three serial passages and serotyped using the standardized methods for Salmonella.

Antimicrobial susceptibility test

The susceptibility against OEO and ENR of strains of the MSW was tested using the broth microdilution method according to the CLSI (2015). Microtiter plates (Greiner Bio-one, Barcelona, Spain) with twofold dilutions of each antimicrobial agent, at concentrations ranging from 10,000 to 78.125 μg/mL for OEO and from 4 to 0.03 μg/mL for ENR, were incubated with a bacterial suspension of 5 × 10⁵ CFU/mL.

Molecular study

The QRDR of the original and selected strains with reduced susceptibility was amplified by polymerase chain reaction (Ruiz-Barba et al., 2005; Dahiya et al., 2014). Nucleotide sequencing was performed by the Central Research Support Service (SCAI) of the University of Cordoba, Spain, using the dye chemistry ABI Big-Dye 3.1 protocol and an ABI 3130XL Genetic Analyzer. Sequences were obtained for all the QRDRs using as reference the sequence LT2 of Salmonella Typhimurium strain (accession No. NC-003197.2 GenBank, Reference Sequence: AE-006468.2). The genes were identified as follows: gyrA: 1253794 (c2373710-2376422); gyrB: 1255362 (c4038868-4041293); parC: 1254697 (c3336954-3339227); parE: 1254704 (c3343969-3345861). The sequence chromatography files were analyzed using Sequencing Analysis v.5.2.0 to resolve the ambiguities of the nucleotides. There are no previous studies about the possible genetic resistance of Salmonella spp. to OEO against which to verify the results.

Results

The broth microdilution MIC and MPC values, as well as MPC/MIC ratios, are described in Table 2. Below we present the results for each of the tests carried out.

Table 2.

Minimum Inhibitory Concentration, Mutant Prevention Concentration, and Mutant Prevention Concentration/Minimum Inhibitory Concentration Index of Original Fully Susceptible Strains and Submutant Prevention Concentration Selected Strains (Induction Test)

Strains	Selected strains	MIC (μg/mL)	MPC (μg/mL)	MPC/MIC
OEO
Original strains
ATCC 14028	1	312.5	625	2
54UCO001	1	312.5	625	2
57UCO003	0	312.5	NMPC	None
Induction test^a
ATCC 14028 1 ×	None	312.5	NMPC	None
54UCO001 1 ×	None	312.5	NMPC	None
Enrofloxacin
Original strains
ATCC 14028	2	0.0625	0.25	4
54UCO001	2	0.0625	0.25	4
57UCO003	2	0.0625	0.25	4
Induction test^a
ATCC 14028 1 ×	None	0.125	NMPC	None
54UCO001 1 ×	None	0.125	NMPC	None
57UCO003 1 ×	None	0.125	NMPC	None
57UCO003 2 ×	None	0.25	NMPC	None

QRDR mutations study of original strains and induction test strains showed in all cases the wt of the genes gyrA, gyrB, parC, and parE, ruling out the presence of mutations.

1 × and 2 × , strains with growth in plates supplemented with a drug concentration of 1 × or 2 × MIC, respectively.

MIC, minimum inhibitory concentration; MPC, mutant prevention concentration; NMPC, no MPC; OEO, oregano essential oil; QRDR, quinolone resistance determining region; wt, wild type.

Oregano EO

The concentration necessary to completely prevent the growth of the bacterial population (MPC) was 625 μg/mL (MPC/MIC = 2) for all isolates except 57UCO003, which only showed growth at concentrations <MIC (78.125–156.25 μg/mL). For the remaining strains (ATCC 14028 and 54UCO001), isolates were recovered from MHA plates containing 78.125–312.5 μg/mL (1/4 × –1 × MIC). However, MIC values of these isolates were similar to those of original strains (312.5 μg/mL).

Enrofloxacin

The ENR MPC was 0.25 μg/mL for three strains (MPC/MIC of 4). This value was lower than the ENR resistance breakpoint (≥2 μg/mL) (CLSI, 2015). Isolates were recovered from MHA plates with 0.0625 and 0.125 μg/mL (1 × and 2 × MIC), showing a decreasing number of colonies as the concentration of antibiotic increased. The MIC of these isolates (0.125–0.25 μg/mL) was higher than the MIC of corresponding original susceptible strain (0.0625 μg/mL), suggesting a reduction in susceptibility to ENR, although the resistance breakpoint was not reached.

Single-step mutation study

The characterization of QRDR of original strains and those produced as a result of the induction test showed in all cases the wild type (wt). The results of OEO resistance induction test for possible “one step” mutations demonstrated the growth of Salmonella Typhimurium strains at concentrations higher than MIC. Unlike OEO, the ENR induction test showed a reduction in the susceptibility of wild strains that allowed its growth at 1 × and 2 × MIC concentrations, resulting in MPC of four times this value. The absence of genetic mutations in QRDR of these strains would support the conclusion that other resistance mechanisms were involved.

Discussion

We thought that it would be convenient to make a first assessment on the possible induction of strains with reduced susceptibility to OEO by mutation, since this is an important mechanism in the resistance of this bacterial species to fluoroquinolones.

In vitro tests based on the determination of MIC against standardized bacterial inoculum (10⁵ CFU/mL) have been the basis of susceptibility tests for decades, as well as the guide for the management of infections. Nevertheless, the correlation between in vitro susceptibility and clinical results is not always 100%, because of other factors, such as the host immune response and the possible presence of resistant bacterial subpopulations (Blondeau, 2009). As a result, many authors highlight difficulties in controlling serious infections with a high bacterial density, probably due to the development of mutations in the bacterial genome during replication, and propose the determination of MPC as a new in vitro measure to complete susceptibility studies (Dahiya et al., 2014; Nordqvist et al., 2016). The antibiotic of choice would therefore be one capable of maintaining a concentration in the target tissue equal to or higher than the MPC for sufficient time to allow it to act against the sensitive and resistant subpopulations, and without exceeding in any case the toxic dose for the organism (Pasquali and Manfreda, 2007; Balaje et al., 2013).

This study, conducted to assess possible resistance to OEO due to single-step mutations, determined for the strains tested MPC values twice the MIC. These results, like those described by Budzyńska et al. (2013), seemed to suggest a possible reduction in the susceptibility of the strains to EOs, which could have been seen in the results of the strain 57UCO003 that did not present MPC if had exhibited growth of the strains for the induction test. However, MIC values obtained in the susceptibility test performed in our work with the strains of the induction test were the same as those of the original strains, ruling out a priori the development of resistance to the OEO.

Other investigations in this field have suggested the implication of general resistance mechanisms such as the efflux pump. The works of Papadopoulos et al. (2008), with tea tree EOs and its main components, showed a reduction in the susceptibility of Pseudomonas aeruginosa to block or genetically modify the multidrug system of active expulsion MexAB-OprM. These findings were based on at least a fourfold increase of the MIC of modified strains with respect to wild ones.

Among the advantages described by many authors regarding the use of EOs is their complex chemical composition and their ability to alter bacterial survival by various action mechanisms, which decrease the presence of new resistant strains (Nazzaro et al., 2013; Knezevic et al., 2016). In OEO, the main components (thymol and carvacrol) may act synergistically increasing the antimicrobial activity of the EO against Gram-positive bacteria. However, thymol alone presented an antibacterial activity similar to EOs against P. aeruginosa, with no statistical difference when compared with EOs (Veras et al., 2012).

For the development of this study, ENR was used as quality control. Fluoroquinolones are the antimicrobials most used today in human and animal medicine due to their broad spectrum and their physicochemical properties. The use of quinolones in veterinary medicine is considered a matter of special public health concern (FDA, 2013; EFSA, 2015; ECDC, 2016; OIE, 2016), since it could contribute to the acquisition of resistance in bacteria transmitted by food such as Salmonella spp., Campylobacter spp., and E. coli, which, in turn, could lead to a reduction in efficacy of the treatment of these infections in humans (Köck and Kreienbrock, 2017).

The results obtained in this study showed a reduction in the susceptibility of the wt, with growth at concentrations 1 × and 2 × MIC and an MPC of four times this value, although in none of the cases could the strains resulting from the induction be considered resistant with the effective concentration remaining below the cutoff point (0.25 μg/mL). Similar results have been described by Pasquali and Manfreda (2007) and Randall et al. (2004), who reported an increase in effective concentration of ENR of 4–64 times the MIC of the wild strains—concentrations that were both lower and higher than the cutoff point (0.5–4 μg/mL). As in these studies, the molecular study of the strains with reduced susceptibility did not show mutations in QRDR. Although the appearance of mutations in the target genes is considered the main resistance mechanism of Salmonella Typhimurium against fluoroquinolones, the difficulty found in inducing such mutations confers increasing relevance on active mechanisms (Pasquali and Manfreda, 2007).

The induction of mechanism of active expulsion (efflux pump) as a result of exposure to high concentrations of antimicrobial, to which other research referred (Pasquali and Manfreda, 2007; Marchetti et al., 2011), could explain the reduction observed in our work in the susceptibility of the strains resulting from the induction. This mechanism alone has a limited effect on resistance, although it is considered that these bacteria will better survive the pressure of antimicrobials and could develop subsequent genetic mutations (Rodríguez-Martínez et al., 2007; Marchetti et al., 2011).

The final approval of EOs as drugs will require, among other things, additional studies on their pharmacokinetic and pharmacodynamic properties, in vivo effects and possible emergence of tolerance and resistance. However, there are few studies on the emergence of resistance to EOs by genetic mutations.

Conclusion

The selective pressure of sensitive Salmonella Typhimurium strains from OEO and ENR produced the growth of isolates above the MIC values, although only in the case of ENR was this accompanied by a nonmutational reduction in antimicrobial susceptibility. This result supports the need to consider new parameters such as the MPC in the determination of the dosage guidelines for ENR, and the possibility of using the OEO as an alternative in the control of salmonellosis. However, in vitro studies with more clinical strains are needed to support their inclusion in salmonellosis surveillance programs.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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