Antimicrobial potential of Pectis substriata essential oil (Asteraceae) against drug-resistant Staphylococcus strains

: Resistant bacterial infections represent one of the major threats in worldwide health services. In this scenario, plant essential oils are considered promising antimicrobial agents. Therefore, this study aimed to evaluate the antimicrobial potential of Pectis substriata essential oil alone and in combination with antibiotics, against clinical drug-resistant bacterial strains. The essential oil from the plant aerial parts was obtained by hydrodistillation. Antimicrobial activity was assessed against standard and clinical bacterial strains by broth microdilution method, and the synergistic effect was evaluated by checkerboard microtiter assay. The oil alone showed signifi cant activity against clinical Staphylococcus warneri (62.5 µg.mL -1 ), and was moderately active on Staphylococcus aureus (standard strain) and clinical Staphylococcus intermedius (125 and 250 µg.mL -1 , respectively). Synergism was achieved for the combinations of essential oil and ampicillin on S. warneri and of oil and kanamycin on S. intermedius . Additive effects were also observed. This is the fi rst report of the chemical composition of P. substriata essential oil, and the results revealed the presence of compounds with proven antimicrobial activity. The oil proved active against resistant Gram-positive cocci, and showed synergism with antibiotics, revealing its potential use as adjuvant or in the development of new alternative treatments of drug-resistant


INTRODUCTION
The antibacterial activity of plant essential oils is well documented, and they are described as promising antimicrobial agents (Simões et al. 2009). Recently, their potential has been exploited alone, or in combination with antibiotics, acting as sensitizers or targeting virulence factors, through synergistic effect (Valdivieso-Ugarte et al. 2019, Yu et al. 2020. Synergic effect consists in the biological action triggered by the combination of two or more chemical entities in the treatment of a pathology, in which the activity of a combination of compounds is higher than the sum of the effects of individual compounds (Efferth & Koch 2011, Tyers & Wright 2019. The main mechanisms by which synergy effects are achieved consist in multitarget actions, such as increasing bioavailability, inhibiting or suppressing antibiotic resistance, particularly leading to the use of concentrations above the minimum inhibitory concentration (MIC), also contributing with the reduction of possible adverse effects (Wagner & Ulrich-Merzenich 2009, Yang et al. 2014, Shin et al. 2018). In addition, avoiding the use of fi rst line therapy drugs with a broad spectrum of antibacterial action, and re-sensitizing the bacteria for the most common types of antibiotics, lessens the evolutive pressure and, consequently, antibiotic resistance. Decreasing the amount of antibiotics via synergistic effect also has the advantage to show less cytotoxicity to human cells and symbiotic gut bacteria. Since the microbiota is closely related to the immune system, the ability to preserve this microbiota is considered a therapeutic priority (Belkaid & Hand 2014).
Plant natural products are considered an important source of bioactive molecules, due to the wide array of complex and structurally diverse molecules produced by these organisms, with great pharmacological potential. Brazilian flora, enclosed in biomes such as Pantanal and Cerrado, represents one of the highest potentials in terms of biological diversity. The Pantanal, considered the word largest wetland´s area, is distributed among the countries of Bolivia, Paraguay, and Brazil, which houses about two-thirds of its area. Although several bioactive molecules have been identified from Pantanal plants (Garcez et al. 2016), the rich flora of this biome still offers a wide biodiversity of species that remains unexplored. Therefore, the Pantanal consists of a promising source of natural products to be investigated regarding their potential synergistic effects in combination with routinely used antibiotics for the treatment of bacterial infections.
Plants belonging to the genus Pectis (Asteraceae) attract attention due to their pronounced fragrances, a characteristic trait observed in other genera in Tageteae tribe. The aromatic properties of Pectis species have been associated with their use in folk medicine, such as P. stenophylla, from which an infusion of the aerial parts is prepared and the hot vapor is inhaled to treating colds (Gentry 1963), and P. prostrata, used for colds and tuberculosis (Austin 2004). Plants rich in essential oil are often appreciated for their use for flavoring food, such as P. elongata (Maia et al. 2005) and P. brevipedunculata (Marques et al. 2013), or in perfumery, such as P. angustifolia (Bradley & Haagen-Smit 1949).
Although Pectis species are renowned for their noticeable fragrances and are widely distributed in the Americas, most of the members of this large genus, comprising c. 90 species (Hansen et al. 2016), have not yet been investigated or are still poorly explored. To the best of our knowledge, the essential oil from Pectis substriata has not been previously investigated.
Once antimicrobial resistance is a severe public health problem and a growing phenomenon, for which combat strategies are urgently needed, we aimed to evaluate the antimicrobial potential of the essential oil against a panel of clinical resistant bacterial strains, alone and/or in combination with known antimicrobial agents, and to report for the first time the chemical composition of the essential oil of the aromatic plant P. substriata from Pantanal biome, Brazil.

Essential oil extraction
Fresh aerial parts (leaves and stems) of P. substriata (1.030 kg) were extracted for 5 hours using a Clevenger-type apparatus, to yield 4.2 g (yield of 0.41% m/m) of essential oil.

Chromatography conditions
T h e e ss e n t i al o i l was d i ss o lv e d i n dichloromethane (1 mg.mL -1 ) and an injection volume of 1 μL was employed, with a split ratio of 1:50. Injector temperature was 250°C, with the carrier gas (Helium 99.999% purity) at a flow rate of 1 mL.min -1 , and pressure of 87.1 kPa. The oven temperature was programmed from 50°C (isothermal for 1.5 min), with an increase of 3°C/ min, to 260°C, ending with a 5 min isothermal at 260°C. A mixture of linear hydrocarbons (C9 to C22 alkanes) was injected under the same experimental conditions. The identification of the constituents in the essential oil was performed by comparing the mass spectra obtained with those of the equipment database (Wiley 7 lib and Nist 08 lib) and by using the Retention Index (RI), calculated for each constituent as previously described (Adams 2017).

Antimicrobial susceptibility assays
All reagents and media for the antibacterial assays were purchased from Sigma Aldrich™. The bacterial strains used were Staphylococcus aureus (NEWP0023, sensitive to amoxicillin, amoxicillin + clavulanic acid, gentamicin, cephalexin, cefoxitin, streptomycin and azithromycin), Escherichia coli (NEWP0022, sensitive to amoxicillin, amoxicillin + clavulanic acid, gentamicin, cephalexin, cefoxitin, streptomycin and tetracycline), both purchased from NEWPROV™ Company, clinical S. aureus (from human intra-abdominal fluid, β-lactamase producer, mecA mediated methicillin resistance), clinical S. warneri (from human hemoculture, β-lactamase producer, resistant to ampicillin, erythromycin and tetracycline) and clinical S. intermedius (from ulcerated nodule on dog skin, mecA mediated methicillin resistance, resistant to amoxicillin + clavulanic acid, gentamicin, neomycin, azithromycin, cephalexin, cephalothin, streptomycin and marbofloxacin). Human clinical strains were provided by the Center of Clinical Analysis of the University Hospital, Universidade Federal de Mato Grosso do Sul (Campo Grande, Brazil), while the veterinary strain was furnished by the Faculty of Veterinary Medicine and Animal Science of Universidade Federal de Mato Grosso do Sul. The antimicrobial activity of the essential oil alone was determined by broth microdilution method, as described by Manda et al. (2018). Two-fold dilutions were performed in 96-well plates prepared with Mueller-Hinton broth to reach a final concentration of 31.3 μg.mL -1 to 4000 μg.mL -1 , with a 100 μL final volume in each well. The inoculums were overnight cultures of each bacterial species in Mueller-Hinton agar diluted in sterile saline solution (0.45%) to a concentration of approximately 10 8 CFU.mL -1 (0.5 in McFarland scale), measured in a MS Tecnopon MCF-500 McFarland turbidimeter. This solution was diluted 1/10 in saline solution (0.45%) and 5 μL were added to each well containing the test samples. All experiments were performed in triplicate and the microdilution trays were incubated at 36°C for 18 h. Then, 20 μL of an aqueous solution (0.5 %) of triphenyl tetrazolium chloride (TTC) were added to each well and the trays were again incubated at 36°C for 2 h. In those wells where bacterial growth did occur, TTC changed from colourless to red. MIC was defined as the lowest concentration of each substance at which no colour change occurred and was expressed in μg.mL -1 .
Synergistic interactions were evaluated using the checkerboard microtiter test, following the method described by Solarte et al. (2017). Serial two-fold dilutions of the essential oil (EO) were made vertically in 96-well plates prepared with Mueller-Hinton broth, to reach a concentration of 31.3 μg.mL -1 to 4000 μg.mL -1 , with a 50 μL final volume in each well. Aliquots (50 μL) of antibiotics solutions in Mueller-Hinton broth were added in each well, so the final concentrations varied horizontally from 100 to 0.05 μg.mL -1 . For EO, the final concentrations varied from 15.6 μg.mL -1 to 2000 μg.mL -1 . Bacterial inoculums were prepared as mentioned above, and 5 μL were added to each well containing the test samples, then the plates were incubated at 36°C for 18 h. After addition of TTC, MIC of the combinations were accessed, and fractional inhibitory concentration (FIC) and fractional inhibitory concentration index (FICI) were calculated by the formulas:

RESULTS AND DISCUSSION
The essential oil of P. substriata (EO) was first evaluated against Staphylococcus aureus (NEWP0023) and Escherichia coli (NEWP0022), two sensitive standard strains, by broth microdilution assay, and showed antimicrobial activity only against S. aureus, with a minimum inhibitory concentration (MIC) of 125 µg.mL -1 .
This selectivity encouraged us to select a panel of clinical Gram-positive strains, with different sensitivity profiles to antibiotics. The chosen strains were Staphylococcus aureus (human pathogen, methicillin mecA-mediated resistance, β-lactamase producer), S. warneri (human pathogen, β-lactamase producer) and S. intermedius (canine pathogen, methicillin mecA-mediated resistance, resistant to penicillins, aminoglycosides, first-generation cephalosporins and quinolones).
Staphylococcus microorganisms are important targets, as they have a diverse arsenal of pathogenicity factors, represented by adhesins, enterotoxins, hemolysins, leukocidins, biofilm production, ability to invade epithelial cells, among others, which contribute to colonize and damage their hosts (da Costa et al. 2011). S. aureus is the major pathogen causing bacterial infection in community settings and hospitals, significantly contributing to morbidity and mortality, as it has the capability to generate a diverse array of infection in different organs or tissues, including skin wound infection, folliculitis, pneumonia, endocarditis, and bacteremia (Yeh et al. 2020). S. warneri is a coagulase-negative common commensal colonizing human and animals' skin and mucosal membranes, that are gaining clinical attention. This bacteria can cause sepsis (e.g. in immunocompetent patients with multiple abscesses), infections related to communityacquired native valve endocarditis, it was also detected in orthopaedic cases, and may be responsible for urinary tract infections (Liu et al. 2020, Szemraj et al. 2020. S. intermedius is a bacterial strain commonly found in skin and mucosal flora in a variety of animals, including dogs and cats, but rarely isolated from humans (Wang et al. 2013). Once antimicrobial resistance genes are very promiscuous, circulating through humans, animals, and the environment, the transmission of resistance from animal microorganisms by direct contact between humans and animals is a concern (Wegener 2012). Therefore, the initiatives working within the One Health concept (Nyatanyi et al. 2017), including animal pathogens in the research for new antimicrobial drugs, can be seen as a more interesting strategy to mitigate the problem of antibiotic resistance.
The oil was tested alone and in combination with antimicrobial drugs against resistant bacteria, in order to check for possible synergistic interactions. Results can be seen in Table I. The P. substriata EO alone showed a significant activity against the human pathogen S. warneri (MIC of 62.5 µg.mL -1 ), and was moderately active on S. intermedius (MIC of 250 µg.mL -1 ) (Wamba et al. 2018). Among all staphylococcal strains assayed, only S. warneri is a coagulase-negative, characteristic that may be related to the enhanced EO activity against this strain. The coagulase enzyme is considered an indicator of virulence, and most of S. aureus and S. intermedius are coagulase-positive (da Costa et al. 2011).
The combination effect of the EO and the antibiotics against resistant bacteria were also evaluated. The results can be seen in Table I. All strains evaluated were β-lactamase producers, hence the combination of ampicillin and the EO was tested for all of them. The EO had additive effects on S. aureus and S. intermedius (FICI=1), while synergism was observed for S. warneri (FICI=0.25). Especially for this antibiotic, a remarkable decrease in the MIC values, from >100 to 0.049 µg.mL -1 , was observed for all tested strains. In combination with tetracycline, the oil had an additive affect when tested for S. warneri, with FICI of 0.75, lowering the antibiotic MIC from >100 to 25 µg.mL -1 . Four-fold reduction in MICs of kanamycin and EO were observed when assayed in combination against S. intermedius, with FICI of 0.5, reflecting synergism.
Essential oils from aromatic plants, such as cinnamon, clove, oregano, lavender and thyme, have shown synergistic effects with antibiotics Table I. Minimum inhibitory concentration (MIC, in µg.mL -1 ) of antibiotics and Pectis substriata essential oil, fractional inhibitory concentration (FIC) and fractional inhibitory concentration index (FICI), of antibioticsessential oil pairs, against drug-resistant bacteria.  The analysis and identification of thirty compounds, representing 98.91% of total oil constitution, was performed by Gas Chromatography coupled to Mass Spectrometry (GC/MS). The essential oil from the aerial parts of P. substriata was composed of monoterpenes and aliphatic compounds, with a single component, perillaldehyde, performing 62.15% of the total oil, followed by 4-undecanol (12.05%), limonene (6.46%), α-fenchene (2.89%) and perillyl alcohol (2.51%) (Table II, Supplementary Material - Figure  S1, Table SI).
A similar result was observed in the analysis of the chemical composition of an Amazonian specimen of P. elongata Kunth leaf oil, which was also characterized by the presence of perillaldehyde (51.7%) and limonene (43.7%) as main compounds (Maia et al. 2005). The monoterpene perillaldehyde has also been defined as the main component of the essential oil from the aerial parts of the Cuban species P. floribunda Kunth and P. prostrata, representing 44.5% and 70.7% of the total leaf oil, followed by limonene, with values of 9.7% and 16. 2%, respectively (Pino et al. 19992%, respectively (Pino et al. , 1996, as well as in the composition of P. odorata, collected in Paraguay, which is rich in perillaldehyde and thymol (Hirschmann et al. 1986).
The essential oil of plants belonging to Pectis genus, such as Pectis brevipedunculata, have shown bactericidal and fungicidal activities (Marques et al. 2013). Although the antimicrobial effects of the P. substriata have not been previously investigated, the main compounds found in the essential oil of this species are known to be biologically active and exhibit antimicrobial, anticancer, and antiinflammatory properties (Duelund et al. 2012). Perillaldehyde efficiently inhibits airborne microbes using an air-washer, contributing to improve environmental health (Sato et al. 2006), and showed antibacterial activity against respiratory tract pathogens, such as Haemophilus influenza, Streptococcus pneumoniae, S. pyogenes [minimum inhibitory dose (MID) of 12.5 mg.L -1 air, respectively], and Staphylococcus aureus (MID=50 mg.L -1 air) (Inouye et al. 2001), and against foodborne bacteria Escherichia coli, E. coli 0157:H7, Salmonella typhimurium (MIC=500 µg.mL -1 , respectively), and Vibrio vulnificus (MIC=250 µg.mL -1 ) (Kim et al. 1995). Perillyl alcohol proved to be a potent natural chemosensitizing agent against fungal infections, acting by multiple mechanisms of action (Ansari 2016), and has a strong antibacterial effect against the periodontal pathogens Fusobacterium nucleatum and Porphyromonas gingivalis (Figueiredo et al. 2020), while (+)-limonene has been described as a bactericidal agent to be used in food preservation, effecting Gramnegative bacteria (Espina 2013). The presence of compounds with proven antimicrobial activity reinforces the potential pharmacological use of essential oils, as well as preservative and flavoring agents. This is the first study regarding the biological activity and chemical composition of Pectis substriata essential oil. The use of combinations that have a synergistic or additive antimicrobial effect is an interesting approach, and our results reinforce that essential oils play a prominent role in this scenario. The use of adjuvant antibacterial agents decreases the amount of antibiotics used, therefore decreasing their Manuscript received on April 6, 2020; accepted for publication on October 7, 2020