The Potential of Use Basil and Rosemary Essential Oils as Effective Antibacterial Agents

The considerable therapeutical problems of persistent infections caused by multidrug-resistant bacterial strains constitute a continuing need to find effective antimicrobial agents. The aim of this study was to demonstrate the activities of basil (Ocimum basilicum L.) and rosemary (Rosmarinus officinalis L.) essential oils against multidrug- resistant clinical strains of Escherichia coli. A detailed analysis was performed of the resistance of the drug to the strains and their sensitivity to the tested oils. The antibacterial activity of the oils was tested against standard strain Escherichia coli ATCC 25922 as well as 60 other clinical strains of Escherichia coli. The clinical strains were obtained from patients with infections of the respiratory tract, abdominal cavity, urinary tract, skin and from hospital equipment. The inhibition of microbial growth by both essential oils, presented as MIC values, were determined by agar dilution. Susceptibility testing to antibiotics was carried out using disc diffusion. The results showed that both tested essential oils are active against all of the clinical strains from Escherichia coli including extended-spectrum β-lactamase positive bacteria, but basil oil possesses a higher ability to inhibit growth. These studies may hasten the application of essential oils in the treatment and prevention of emergent resistant strains in nosocomial infections.


Chemical Composition of the Basil and Rosemary Essential Oils
Chemical analysis showed the presence of forty-eight constituents within basil oil from Ocimum basilicum L., the main ones being estragole (86.4%), 1,8-cineole (4.9%) and trans-α-bergamotene (3.0%). The chemical composition of the basil oil is presented in Table 1. The essential oil from Rosmarinus officinalis L. contains thirty-seven components: the main ones being 1,8 cineole (46.4%), camphor (11.4%), α-pinene (11.0%), β-pinene (9.2%) and camphene (5.2%). The chemical composition of the rosemary oil is presented in Table 2.  Extended spectrum β-lactamase production for the tested Escherichia coli clinical strains was detected for strains from the abdominal cavity (n = 4), bronchia (n = 4), wounds (n = 4), urine (n = 4) and for strains isolated from blood (n = 3) and catheters (n = 3). The results are shown in Figure 1. The tested strains of Escherichia coli (ESBL+) were generally resistant to β-lactams, aminoglycosides and quinolones recommended for susceptibility testing. Most of them were resistant to cephalosporins and β-lactam antibiotics with such inhibitors as clavulanic acid, sulbactam and tazobactam.

The Susceptibility Escherichia coli Bacterial Strains to Basil Oil
The MIC values for sixty tested E. coli strains were between 8.0 µL/mL to 11.5 µL/mL. The basil oil showed inhibitory activity against E. coli ATCC 25922 standard strain at 8.0 µL/mL.

The Susceptibility Escherichia coli (ESBL+) Strains to Basil Oil
Most E. coli ESBL+ strains isolated from the abdominal cavity (n = 4) and from the bronchia (n = 4) were sensitive to basil oil at a concentration range from 8.25 µL/mL to 9.0 µL/mL. For (ESBL+) clinical strains from wounds (n = 4), MIC values were between 8.5 µL/mL to 9.25 µL/mL. The growth inhibition concentrations for bacteria isolated from blood were 8.75 µL/mL (n = 1) and 9.25 µL/mL (n = 2). Basil oil at a concentration of 8.75 µL/mL inhibited the growth of one of the tested clinical strains isolated from urine, while 9.0 µL/mL inhibited the growth of three. The MIC values for strains from catheters were 8.75 µL/mL (n = 2) and 9.25 µL/mL (n = 1). The susceptibility of Escherichia coli (ESBL+) strains to basil oil is shown in Figure 3.

The Susceptibility Escherichia coli (ESBL−) Strains to Basil Oil
The basil oil was active against Escherichia coli (ESBL−) strains at a concentration range from 8.25 µL/mL to 11.5 µL/mL. The most tested strains isolated from the abdominal cavity (n = 4) were inhibited by concentrations of 11.0-11.5 µL/mL. Clinical strains from the bronchia (n = 5) were sensitive to basil oil at concentrations of 10.25-11.5 µL/mL. The MIC values for most strains from wounds (n = 4) were from 10.0 µL/mL to 11.5 µL/mL. Most Escherichia coli (ESBL−) strains isolated from blood and urine were inhibited by basil oil at concentrations ranging from 9.5 µL/mL to 11.5 µL/mL. The MIC values for bacteria from the catheters (n = 6) were 10.0-11.5 µL/mL. The susceptibility of Escherichia coli (ESBL−) strains to basil oil is demonstrated in Figure 3.

The Susceptibility Escherichia coli Bacterial Strains to Rosemary Oil
The rosemary oil was less active against the sixty tested Escherichia coli clinical strains obtained from the diverse clinical materials and the hospital equipment. The MIC values were between 18.0 and 20.0 µL/mL. The standard strain E. coli ATCC 25922 was sensitive to rosemary oil at a concentration of 18.5 µL/mL.

The Susceptibility of Escherichia coli (ESBL+) Strains to Rosemary Oil
Most Escherichia coli (ESBL+) strains isolated from the abdominal cavity (n = 3) were sensitive to rosemary oil at concentrations from 18.0 µL/mL to 18.5 µL/mL, and (n = 1) at 19.25 µL/mL concentration. The MIC values for bacterial strains isolated from the bronchia (n = 4) and wounds (n = 4) were 18.25-19.0 µL/mL and 18.5-19.25 µL/mL, respectively. E. coli (ESBL+) strains isolated from blood were sensitive to rosemary oil at concentrations between 18.75 and 19.75 µL/mL. All strains from urine (n = 4) were inhibited in the concentration ranges 18.0 µL/mL to 18.75 µL/mL, while those from catheters (n = 3) were inhibited from 18.25 µL/mL to 18.75 µL/mL. The results of the tests detailing the susceptibility of Escherichia coli (ESBL+) strains to rosemary oil are presented in Figure 4.

The Susceptibility of Escherichia coli (ESBL−) Strains to Rosemary Oil
Rosemary oil was found to have MIC values from 18.5 µL/mL to 19.75 µL/mL for isolates from the abdominal cavity (n = 6). Growth inhibition was found to occur at concentrations of 19.0-20.0 µL/mL for clinical bacterial strains obtained from the bronchia (n = 6). Rosemary oil at concentrations ranging from 18.25 µL/mL to 20.0 µL/mL inhibited the growth of all tested Escherichia coli (ESBL−) strains isolated from wounds. For the blood (n = 7) and urine (n = 6) isolates, the MIC values were in the range 18.25-19.75 µL/mL. Finally, similar MIC values were found (18.25-19.75 µL/mL) against E. coli (ESBL−) strains isolated from catheters (n = 7). The susceptibility of the Escherichia coli (ESBL−) strains to rosemary oil is shown in Figure 4.

Discussion
In our investigation, all clinical strains of Escherichia coli were found to be sensitive to basil (Ocimum basilicum L.) and rosemary (Rosmarinus officinalis L.) essential oils, irrespective of the clinical conditions they were obtained under or the pattern of antibiotic resistance they demonstrated, but basil oil was more active against the tested bacteria. Out of the sixty clinical strains of E. coli, twenty-two strains were extended-spectrum beta-lactamase positive (ESBL+). Concentrations of basil oil ranging from 8.25 µL/mL to 9.25 µL/mL were seen to inhibit the growth of eighteen Escherichia coli (ESBL+) strains. For the extended-spectrum beta-lactamase negative strains, seventeen out of the thirty-eight were sensitive to basil oil at concentrations between 11.25-11.50 µL/mL. The MIC values for the other twenty-one Escherichia coli (ESBL−) strains ranged from 8.0 µL/mL to 11.0 µL/mL.
The rosemary oil demonstrated significantly lower activity. No apparent differences in activity of the essential oil were found against extended-spectrum β-lactamase-positive and negative strains. Out of the twenty-two clinical strains of E. coli (ESBL+), rosemary oil concentrations ranging from 18.0 µL/mL to 19.0 µL/mL were effective for eighteen of them. Eleven of the Escherichia coli (ESBL−) strains out of the thirty-eight obtained under various conditions were inhibited by 19.75 µL/mL of rosemary oil. MIC values ranging from 18.25 µL/mL to 19.5 µL/mL for comparable numbers of strains was obtained.
The results of our tests clearly demonstrate that basil and rosemary essential oils can be widely used to eliminate clinical strains of Escherichia coli found in different clinical conditions. It is also significant that extended-spectrum β-lactamase (ESBL)− producing clinical strains of E. coli are sensitive to these oils. Studies Orhan at al [29], confirm that, essential oils from Foeniculum sp., Mentha sp., Ocimum sp., Origanum sp. and Satureja sp. (Lamiaceae family) possess strong antibacterial activity against extended-spectrum beta-lactamase (ESBL) positive strains of Klebsiella pneumoniae isolated from food. The obtained MIC values ranged from 32 to 64 μg/ml for a number of strains resistant to trimetoprime-sulfametoxazole, sulbactam-ampicilin, clavulonate-amoxicilin, ceftriaxon, cefepime, imipenem, ceftazidime, tobramicine, gentamisine, ofloxacin, and ciprofloxacin. According our investigations, the Escherichia coli (ESBL+) responsible for human infectious diseases were significantly more resistant to the basil and rosemary essential oils.
The antimicrobial properties of essential oils are strictly connected with their chemical composition. The usefulness of essential oils as effective antimicrobial agents can be evaluated only by analysing their individual components. Therefore, a thorough GC-FID-MS analysis of the tested basil and rosemary essential oils was conducted. The composition of the essential oil obtained from Ocimum basilicum L., is as given in the ISO-11043 standard. The composition of the tested basil oil corresponded to required standards, according to which the content of estragole must be higher than 75.0%. The composition of the essential oil derived from Rosmarinus officinalis L. was found to meet the requirements of the European Pharmacopoeia 6 [30] and of the Polish Pharmacopoeia VIII [31] for the nine main components. The content of β-pinene amounted to 9.2% (required 4.0%-9.0%) and limonene to 1.0% (required 1.5%-4.0%). Verbenone was not found among the components of the tested rosemary oil, although EP 6 and the Polish Pharmacopoeia VIII specify its content to be a maximum of 0.4%. For the tested rosemary essential oil, nine of the thirteen main constituents of the oil met the requirements given in the ISO-1342 standard: α-pinene, camphene, myrcene, 1,8-cineole, p-cymene, camphor, bornyl acetate, α-terpineol and borneol.
The highest antibacterial activity is demonstrated by phenolic compounds such as carvacrol, thymol and eugenol. Another effective group of active compounds are alcohols: terpinen-4-ol, γ-terpineol, geraniol, cytronellol, menthol and linalol. Many of them are synthesized by plants from the Lamiaceae family [32]. For instance, the essential oil of Satureja hortensis L. demonstrates high levels of activity. Mihajilov-Kristev et al. [33], showed that essential oil containing mainly carvacrol (67.0%) and γ-terpinene (15.3%) is effective against Gram-negative strains, including Escherichia coli, with MIC values from 0.025 µL/mL to 0.78 µL/mL according to the broth microdilution method. In our study for basil and rosemary essential oils, we obtained significantly higher MIC values. This high activity demonstrated by Satureja hortensis L. essential oil is certainly related to the high content of carvacrol, which is one of the most potent antimicrobial compounds.
The literature reports that basil oil, which contains mainly estragole and linalool, also possesses antibacterial agents which are effective against a variety of Gram-positive and Gram-negative bacteria [34]. According to Saković et al. [35], Ocimum basilicum essential oil possesses antibacterial activity against the human pathogenic bacteria Bacillus subtilis, Enterobacter cloacae, Escherichia coli O157:H7, Micrococcus flavus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, S. typhimurium, Staphylococcus epidermidis and S. aureus. According to the authors, basil essential oil containing 69.3% linalool as a main component possesses antibacterial properties against Escherichia coli O157:H7 with MIC and MBC values of 6.0 µg/mL when assessed by microdilution. The multidrug clinical strains of E. coli tested in this study were more resistant to basil essential oil containing mainly estragole (86.4%).
Results obtained by Sartoratto et al. [36], show that basil oil from Ocimum basilicum containing mainly linallol 32.6% and eugenol 28.1%, and oil from Ocimum gratissimum containing 93.9% eugenol, have a broad spectrum of antibacterial activity against reference strains of Gram-positive, Gram-negative bacteria and Candida albicans. According to the authors, essential oils obtained from these two genera of Ocimum were active against Escherichia coli CCT0547 standard strain with a MIC value of >2 mg/mL according to the microplate method. These results also confirm that oils with active constituents such as eugenol tend to have high antibacterial properties. Our results are slightly higher than those obtained by Sartoratto et al. The MIC values were in the range from 7.92 mg/mL to 11.04 mg/mL for clinical strains of Escherichia coli which were both positive and negative for extended-spectrum beta-lactamase activity.
Nakamura et al. [37], demonstrated that the essential oil from Ocimum gratissimum, with eugenol as its main constituent, possesses antibacterial activity against clinical strains of Escherichia coli ATCC 25922 with MIC-6 mg/mL. The MICs of this essential oil against a group of other Gram-negative bacteria comprising Shigella flexneri, Salmonella enteritidis, Klebsiella sp. and Proteus mirabilis were found to be from 0.3 to 12 mg/mL. The MIC values given in our study were slightly higher than those given by Nakamura et al. Pereira et al. [10], in their investigations, showed that oil from Ocimum gratissimum possesses antibacterial activity against clinical Escherichia coli strains isolated from urinary tract infections. The essential oil was found to be active against about 70% of tested E. coli clinical isolates. The results of the present study demonstrate that a chemotype containing mainly 1,8-cineole, eugenol, methyleugenol, thymol, p-cimene, cis-ocimene and cis-caryophyllene has lower activity.
In our tests, basil oil obtained from Ocimum basilicum, containing mainly estragole (86.4%), inhibited the growth all strains isolated from various clinical materials. Among them were bacteria isolated from urine, which were also extended-spectrum beta-lactamase positive. Our studies confirm that antibacterial activity is possessed by not only basil oil chemotypes with linalool or eugenol as their main components, but also that of Ocimum basilicum, containing mainly estragole. Our research showed that basil essential oil was significantly more effective against all clinical isolates than rosemary essential oil. Similar results were obtained by Hammer et al [38], who studied the antimicrobial activity of basil and rosemary essential oils against Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus, using an agar dilution method. The authors confirmed that basil (Ocimum basilicum) oil is more active against Escherichia coli than rosemary (Rosmarinus officinalis). The MIC values were 0.5 and 1.0% (v/v), respectively.
According to Lopez et al. [39], the oils from Ocimum basilicum and Rosmarinus officinalis have an antibacterial potential against the Gram-positive bacteria Staphylococcus aureus, Enterococcus faecalis and Listeria monocytogenes and against Gram-negative bacteria Escherichia coli, Yersinia enterocolitica, Salmonella choleraesuis and Pseudomonas aeruginosa as foodborne bacterial strains. The authors present a detailed analysis of the tested oils and their ability to inhibit the growth of bacteria. Their basil and rosemary essential oils were of a similar composition to the essential oils in our investigations. The main component of the basil essential oil was estragole-82% ± 1.2%, while the rosemary essential oil contains 1.8-cineole-48% ± 9.1%, camphor-17% ± 4.0, β-pinene-4.8% ± 0.9% and β-caryophyllene-6.8% ± 3.7%. The authors confirm that the basil oil is more effective at inhibiting the growth of Escherichia coli strains.
Probuseenivasan et al [27], confirmed that rosemary essential oil strongly inhibits Escherichia coli ATCC 25922. Although the basil oil was also seen to demonstrate low activity against the tested bacteria, no data was given about the constituents of the essential oils. The minimal inhibitory concentration for rosemary oil against E. coli was >6.4 mg/mL. The MIC values obtained by the present study were higher and ranged from 16.02 mg/mL to 17.35 mg/mL against E. coli clinical strains. Fabio et al [40], report that rosemary oil has an antibacterial effect on a number of microorganisms responsible for respiratory infections, isolated from clinical specimens, among which were antibiotic-sensitive and antibiotic-resistant strains such as Streptococcus pyogenes, S. agalactiae, S. pneumoniae and Klebsiella pneumoniae, Staphylococcus aureus and Stenotrophomonas maltophilia. In our tests, rosemary oil was also found to demonstrate antibacterial activity against Escherichia coli strains with different patterns of resistance, including extended-spectrum β-lactamase positive strains isolated from various clinical materials. The rosemary essential oil used in the present study obtained from Rosmarinus officinalis contains mainly 1,8-cineole (46.4%), camphor (11.4%) and α-pinene (11.0%). The composition of the rosemary essential oil used by Jiang et al. [41], was similar to that used by us: mainly 1,8-cineole (26.54%) and α-pinene (20.14%). The authors show that the tested oil possesses antibacterial activity against Gram-positive bacteria (Staphylococcus epidermidis, Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria (Proteus vulgaris, Pseudomonas aeruginosa and Escherichia coli) and fungi (Candida albicans and Aspergillus niger). Bendeddouche et al [42], showed that of essential oil from Rosmarinus tournefortii De Noé growing wild in the occidental region of Algeria possesses antimicrobial activity also against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) pathogenic bacteria.
A number of studies show that essential oils and their constituents possess useful properties concerning human health. Many of them may be applied in anticancer therapy, cardiovascular and nervous system disorders to reduce the level of cholesterol, to regulate the glucose level or to stimulate hormone production [43]. They also might have great value in preventing and treating infectious diseases. Essential oils not only have bactericidal activity but also can inhibit multidrug bacterial strain formation. Their multiple antibacterial, antifungal, antiviral and also anti-inflammatory and antioxidant effects, have made them valuable agents in human treatment and for the prevention of pathological changes. In addition, essential oils have a number of beneficial properties as natural preservatives in cosmetics, toiletries, drugs and food products [44][45][46]. Considering the huge increase in the number of multidrug resistant bacterial strains in health care facilities, essential oils may prove to be effective natural antimicrobial agents.

Bacterial Strains
The standard strain, E. coli ATCC 25922, was obtained from the collection of the Medical and Sanitary Microbiology Department, Medical University of Lodz. The clinical strains of Escherichia coli were collected in 2011 and 2012 from a range of clinical materials recovered from patients and from the hospital equipment in various wards from one of the Medical University hospitals in Lodz: internal medicine, surgery, urology and the intensive care unit. The tested bacterial strains were isolated from the abdominal cavity (n = 10), bronchia (n = 10), wounds (n = 10), blood (n = 10), urine (n = 10) and from catheters (n = 10).

Bacterial Strain Identification
E. coli strains were cultured on Columbia Agar (bioMerieux, Craponne, France) and on Mac Conkey Agar (bioMerieux). They were identified to the species by using API 20 E tests (bioMerieux). The bacteria were incubated at 37 °C for 24 h.

Essential Oil Analysis
Commercial essential oils from basil-Ocimum basilicum L. and rosemary-Rosmarinus officinalis L. Identification of components was based on the comparison of their MS spectra with those of the laboratory-made MS library, commercial libraries (NIST 98.1, Wiley Registry of Mass Spectral Data, 8th Ed. and MassFinder 3.1) and with literature data [47,48] along with the retention indices on the apolar column (Rtx-1, MassFinder 3.1) associated with a series of alkanes with linear interpolation (C 8 -C 26 ). A quantitative analysis, expressed as percentages of each component, was carried out by peak area normalization measurements without correction factors.

Antibacterial Tests
The standard and clinical strains were cultivated in Columbia Agar medium and incubated at 37 °C for 48 h in aerobic conditions. The microbial suspension was standardized to a cell density of 1-2·× 10 8 cells/mL, equal to an optical density of 0.5 on the Mc Farland scale, by a bioMerieux densitometer. The agar dilution method was employed for the screening of antimicrobial activities of the essential oils [32,38,[49][50][51]. The tested essential oils were diluted in 96% ethanol PURE (POCH, Gliwice, Poland) yielding a concentration of 97% v/v of oils. Although the tested essential oils dissolve well in ethanol, only minimum amounts were used, as it can inhibit the growth of the tested bacteria. This solution was mixed with a culture medium to obtain concentrations from 7.25 µL/mL to 11.75 µL/mL for basil oil and 17.75 µL/mL to 20.25 µL/mL for rosemary oil and poured into sterile Petri dishes. An inoculum containing 1-2 × 10 8 cells/mL (0.1 mL) per spot was seeded upon the surface of the agar with various oil concentrations, as well as on agar with no oil added (acting as a control for strain growth). The Minimal Inhibitory Concentration, MIC, was determined after 24 h of incubation at 37 °C under aerobic conditions. The MIC was considered the lowest concentration of the sample at which no visible growth was observed. The analysis of the antibacterial activity of the oil was performed three times independently. Control media containing only alcohol at concentrations used in the dilutions of tested essential oils did not inhibit the growth of bacterial strains.

Susceptibility Testing
The following antibiotics (Becton Dickinson) were used for susceptibility testing of Susceptibility testing was carried out using the disc-diffusion method, on Mueller-Hinton II Agar (bioMerieux). Cultures were incubated at 37 °C for 16-18 h. The results were interpreted according to EUCAST guidelines [52].
The double-disk synergy test and combination disk method were used to determine ESBL production. The sensitivity of the DDST can be improved by reducing the distance between the disks of cephalosporins and clavulanate. The disk approximation method was performed on a Muller-Hinton agar plate inoculated with the clinical bacterial strain, by placing disks containing CAZ-ceftazidime (30 µg), CTX-cefotaxime (30 µg) and ATM-aztreonam (30 µg) 20 mm (edge to edge) from a disk of AMC-amoxicillin/clavulanic acid (20/10 µg). Following incubation for 16-18 h at 37 °C, any enhancement of the zone of inhibition between a cephalosporin and monobactam-aztreonam disk from the amoxicillin/clavulanic acid disk, was indicative of the presence of an ESBL. E. coli ATCC 25922 was used as a positive control [11,53].

Conclusions
(1) The results of these experiments indicate the potential use of basil and rosemary essential oils against resistant Escherichia coli clinical strains, and also against extended-spectrum β-lactamase positive bacteria. (2) The tested basil oil was more active against all Escherichia coli clinical strains.
(3) The action of essential oils against bacteria exhibiting different mechanisms of resistance may be useful, not only in treating but also preventing the spread of resistant strains.