Antibacterial, antifungal, and antiviral effects of three essential oil blends

Abstract New agents that are effective against common pathogens are needed particularly for those resistant to conventional antimicrobial agents. Essential oils (EOs) are known for their antimicrobial activity. Using the broth microdilution method, we showed that (1) two unique blends of Cinnamomum zeylanicum, Daucus carota, Eucalyptus globulus and Rosmarinus officinalis EOs (AB1 and AB2; cinnamon EOs from two different suppliers) were active against the fourteen Gram‐positive and ‐negative bacteria strains tested, including some antibiotic‐resistant strains. Minimal inhibitory concentrations (MICs) ranged from 0.01% to 3% v/v with minimal bactericidal concentrations from <0.01% to 6.00% v/v; (2) a blend of Cinnamomum zeylanicum, Daucus carota, Syzygium aromaticum, Origanum vulgare EOs was antifungal to the six Candida strains tested, with MICs ranging from 0.01% to 0.05% v/v with minimal fungicidal concentrations from 0.02% to 0.05% v/v. Blend AB1 was also effective against H1N1 and HSV1 viruses. With this dual activity, against H1N1 and against S. aureus and S. pneumoniae notably, AB1 may be interesting to treat influenza and postinfluenza bacterial pneumonia infections. These blends could be very useful in clinical practice to combat common infections including those caused by microorganisms resistant to antimicrobial drugs.


| INTRODUCTION
Antimicrobial resistance poses a serious threat to the effective treatment of an ever-increasing range of infections caused by bacteria, fungi and viruses. Worldwide, antibiotic resistance is increasing.
For example, Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae have reported reduced antibiotic susceptibility, which exceeded 50% in most countries that provided data to the WHO Antimicrobial Resistance Global Report on Surveillance (WHO, 2014).
Candidiasis has also become substantially problematic, with Candida albicans showing increased resistance to common antifungal agents (Goncalves, Souza, Chowdhary, Meis, & Colombo, 2016;Hawser & Douglas, 1995). The recent pandemic of a novel H1N1 influenza viral strain and emerging strains resistant to commonly used antiherpes simplex drugs also emphasizes the need to identify effective approaches to prevent and treat viral infections (Boivin, 2013;James & Prichard, 2014).
This increasing resistance has created a need to develop new antimicrobial agents. Essential oils (EOs) are good candidates as studies have shown that individual EOs and their isolated compounds, including terpenes and terpenoids (1,8-cineole, carvacrol) and aromatic compounds (cinnamaldehyde and eugenol) have antimicrobial activity against a wide range of pathogens, with various spectrums of activity (Bassole & Juliani, 2012;Friedman, Henika, & Mandrell, 2002;Jantan, Karim Moharam, Santhanam, & Jamal, 2008). The antimicrobial effects of EOs are linked to their composition and cytotoxic effects, which cause cell membrane damage. EO compounds are lipophilic, and so pass through the cell wall and cytoplasmic membrane. They disrupt the structure of the polysaccharide, fatty acid, and phospholipid layers, making the membrane permeable (Bakkali, Averbeck, Averbeck, & Idaomar, 2008). Unfortunately, EOs do not specifically target pathogens; they can also affect eukaryotic cells in a reversible or irreversible manner (Carson, Hammer, & Riley, 2006).
In extreme cases, EO cytotoxicity can lead to apoptosis, necrosis, and organ failure (Tisserand & Young, 2013). Therefore, EOs have to be used carefully, within the daily intake limits defined by the relevant authorities when available (EMEA andHMPC 2010, 2011;FAO and WHO 2003).
The antibacterial activity of AB1 and AB2 was evaluated in vitro against a selection of Gram-positive and Gram-negative bacteria, with or without antibiotic resistance, AB1 was evaluated for antiviral activity and AF was assessed for activity against different Candida strains.

| Antibacterial and antifungal assays
Strains were preserved at −80°C and subcultured on (1) (3) Sabouraud agar (Biomérieux) under aerobic conditions at 30°C for yeasts. Suspensions were prepared in sterile distilled water to obtain a final inoculum of 10 8 CFU/ml for bacteria and 10 7 CFU/ml for yeasts.
Blends AB1 and AB2 were tested for their antibacterial activity and AF for its antifungal activity according to a previously reported micromethod (Ibrahim et al., 2012). Tests were also performed with amoxicillin for bacteria and amphotericin B for yeasts as a control for microorganism sensitivity.
Each blend was diluted, using twofold steps in microtiter plates in culture medium: (1)  for H. influenza IP 102514; and (4) Sabouraud (Biomérieux) for yeasts, from column 1 to column 10. Columns 11 and 12 were maintained for sterility control (without product or microorganisms) and growth control (without product and with microorganisms). The twofold dilutions led to emulsions allowing the conduct of tests. Inoculation was performed, using a multipoint inoculator (Denley) under a volume of approximately 1.5 μl for each suspension and microplates were incubated as described above.
Minimal inhibitory concentration (MIC) was defined as the concentration of test compound at which no macroscopic sign of cellular growth was detected in comparison to the control without compound.
It was determined for bacteria after incubation at 36°C for 24 hr and yeasts at 30°C for 24 hr in the presence of serial dilutions of the test All experiments were performed in duplicate at each concentration, using a micromethod analysis based on the CA-SFM guidelines.

| Viral strains and antiviral activity
Antiviral activity of AB1 was tested with influenza A H1N1 ATCC VR-R 1520 and oral herpes simplex HSV1 ATCC VR-1383. Tests were performed according to NF EN 14476 (AFNOR 2015). The H1N1 strain was amplified on MDCK cells (CCL-34, ATCC) and HSV1 on VERO cells in EMEM medium (PAN-Dutscher). Virus suspension was added to the test compound with interfering substance under clean conditions (1% PBS, Sigma Aldrich). This mixture was maintained at 35°C ± 1 for 60 min ± 10. The activity was stopped by the molecular sieving method, using a sieve filter (Sephadex LH 20). Neutralization of the product was validated by passing it through Sephadex at a dilution 1/10.

Virus titration on cells in suspension was performed in microplates.
A dilution series with a factor of four was prepared in an ice-cold medium for 30 min in glass tubes. The dilution was then transferred into microtiter plates before the cell suspension was added in each well. Viral cytopathic effect was read under an inverted microscope after 4 days of incubation and determined by the Spearman-Kärber method (Lorenz & Bogel, 1973)  Reduction in virus infectivity was calculated from the difference of log virus titers before and after treatment. The product was considered to be virucidal when log reduction was ≥4.

Blends AB1 and AB2 were effective against antibiotic-resistant strains
Pseudomonas aeruginosa CIP 103467, Staphylococcus aureus MRSA ATCC 3359, and Escherichia coli ESBL (Table 1). However, P. aeruginosa CIP 103467 was the least sensitive to the blends tested (MBC: 3% v/v for AB2 and 6% v/v for AB1). This result was not surprising as the natural resistance of P. aeruginosa has been previously reported (Longbottom, Carson, Hammer, Mee, & Riley, 2004;Papadopoulos, Carson, Chang, & Riley, 2008). A combination of mechanisms protects this bacteria. The external membrane is particularly impermeable to drugs and has porine-dependent inhibition and efflux mechanisms (Papadopoulos et al., 2008 (Kim, Marshall, & Wei, 1995). However, other studies found EOs were more effective against Gram-positive bacteria or a lack of selectivity for certain EOs (Hammer, Carson, & Riley, 1999;Prabuseenivasan, Jayakumar, & Ignacimuthu, 2006).
On the basis of MBC/MIC ratios, the bactericidal effect was confirmed for AB1 and AB2 for most strains tested (ratios ≤ 2) except for E. coli UTI89 and Y. enterocolitica for the two blends, S. thyphimurium for AB1 and S. pneumoniae and B. fragilis for AB2 (Table 1).
Discrepancies between blends may be explained by the different chemical composition of the two different cinnamon EOs. Although chemotypes of the two cinnamon EOs were the same (CT cinnamadehyde), the cinnamaldehyde concentration in the cinnamon EO was almost twofold higher in AB2 than in AB1 and the eugenol concentration was >30% in AB1 compared to ~2% in AB2.  Wild ™ ), efficacy was shown against H1N1, but was not tested against bacteria (Wu et al., 2010). In our study, AB1 was proven to be effective against both viruses and bacteria in particular, H1N1 virus, S. aureus and S. pneumoniae, two bacteria responsible for postinfluenza pneumonia (Chung & Huh, 2015). This dual activity could be of particular interest to treat influenza and also postinfluenza bacterial pneumonia infections, a leading cause of influenza-associated death.

| Antiviral activity of AB1
This in vitro study shows that blends AB1 and AB2 of C. zeylanicum, D. carota, E. globulus, and R. officinalis EOs possess a highly antimicrobial activity against Gram-positive and Gram-negative bacteria.
Blend AB1 is also effective against viruses. Blend AF-containing C.
zeylanicum, D. carota, S. aromaticum, and O. vulgare EOs had a highly antifungal activity. This suggests that these blends could be effective to combat microorganisms involved in common, acute, and chronic human infections. Further exploration in clinical settings will be needed to confirm these in vitro results in terms of efficacy and also assess their safety.