The antibacterial activity of nano-encapsulated basil and cinnamon essential oils against certain multidrug-resistant bacteria recovered from infected wounds

This study was conducted to evaluate the antibacterial activity of nano-encapsulated essential oils of basil ( Ocimum basilicum ) and cinnamon ( Cinnamomum verum ) against multidrug-resistant (MDR) bacteria. A total of 115 wound swab samples were collected from patients admitted to Naser Institute, Cairo, Egypt, suffering from wounds discharge; pain, swelling, foul-smelling, delayed and non-healing wound infections. Six genera of bacteria were isolated from the collected swab samples, and then identified using conventional biochemical methods and API 20 kits. Staphylococcus aureus was found to be the most prevalent isolate (26.1 %), following by Pseudomonas aeruginosa (25.2 %), Klebsiella pneumonia (23.5 %), Acinetobacter baumannii (12.2 %), Proteus vulgaris (7.8 %), and the less common isolate of Escherichia coli (5.2 %). Among 14 antibiotics tested in vitro for their susceptibility using the standard disk diffusion assay, results showed that imipenem was the most efficient antibiotic against most of the tested Gram (-) and Gram (+) isolates followed by meropenem. Currently, all the recovered bacterial isolates were MDR. The nano-encapsulated basil oil (NEBO) and nano-encapsulated cinnamon oil (NECO) showed potential antibacterial potentials against all the tested MDR bacteria. Results of testing the antibacterial potential of the NEBO and NECO demonstrated that the encapsulation process protected the oils from oxidation, and consequently enhanced their antibacterial potencies. It could be concluded that the nano-encapsulated essential oils act as promising antibacterial agents against the MDR bacteria.


Introduction
The multidrug resistant bacteria (MDR) are defined as those bacteria that cause diseases and withstand the action of the antibacterial drugs, leading to enormous economic and human losses. More than 700,000 people pass away every year all over the world due to the improper use of antibiotics, where the bacterial pathogens develop resistance to the traditional therapy (Willyard, 2017). For example, Staphylococcus aureus which is well known as a methicillin-resistant bacterium caused about 20,000 blood-borne infections, and more than 20,000 correlated deaths in the United States at 2017 (Kourtis et al., 2019). Additionally, Iovleva and Doi, (2017) reported that carbapenem-resistant Enterobacteriaceae was regarded as a public health threat, and requires fast and invasive measures. There are several issues related to the traditional use of antibacterial drugs including diminished stability; low water solubility, drug targeting complexity, minimum oral bioavailability and the compliance of patient depression, due to the frequent administration of drugs and their consequent potential toxicity (Parisi et al., 2017). Recently, the World Health Organization (WHO) reported the increased prevalence of the antibiotic-resistant bacteria, which represents the main human health challenge (Eleraky et al., 2020). The availability of novel antibacterial drugs seems a very complex process, due to the susceptibility of production of new safe and effective drugs, in addition to high costs of production and the long time required for approval of such new drugs (Parisi et al., 2017).
The long term administration of antibiotics generates unbearable toxicity and other adverse effects, and hence, the application of nano-materials may be a promising strategy to control infections of the MDR bacteria (Natan and Banin, 2017;Baptista et al., 2018;Muzammil et al., 2018). Nanoparticles (NPs) have a therapeutic promise due to their unique chemical and physical properties (Beyth et al., 2015;Hemeg, 2017), and are reported to exhibit antibacterial potentials and are able to eliminate growth of the MDR bacteria (Slavin et al., 2017). Nevertheless, the application of NPs to the clinical use needs comprehensive understanding of their physicochemical properties; their pharmacodynamics, pharmacokinetics and bio-distribution (Burdusel et al., 2018). During the past decade, several studies were conducted to discover new antimicrobial essential oils derived from medicinal plants (Rudramurthy et al., 2016), due to their high contents of chemical substances that belong to various families such as aldehydes; terpenes, esters, ethers, alcohol, ketones and phenolics (Akhtar et al., 2014). However, some chemical constitutes of these essential oils loss their activities, due to the changes in their physical and chemical properties caused by the different environmental factors (Abdel-Wahhab et al., 2018). The encapsulation of these essential oils is important to prevent undesirable changes in their components (Arpagaus et al., 2018). Thus, application of nanotechnology is a promising candidate to improve the stability and physicochemical properties of the essential oils, and allows their human use as potent antibacterials against the MDR bacteria (Patra et al., 2018). Therefore, the objectives of the current study were to formulate the encapsulated cinnamon and basil essential oils, and to evaluate their antibacterial efficacies against the MDR bacteria that cause wound infections.

Encapsulation and characterization of the essential oils
Whey protein (WP) recovered from Whey was used at a concentration of 10 % in dist. water as a wall material. The solution was stirred for 1 h, incubated overnight at room temperature, and then Tween 80 was added to the polymer mixture as an emulsifier. The essential oils (cinnamon and basil) were successively added to the polymer solution with homogeneous shaking at 20000 rpm for 10 min. to form an emulsion. The concentration of the polymer was 20 %, and the used amount of the essential oils was 10 % of the mass of the polymer concentration, according to Jinapong et al., (2008). The emulsion solution was encapsulated using the spray drying method in reference to Abdel-Wahhab et al., (2018). The prepared emulsion was spray dried using laboratory Mini spray dryer (BÜCHI, Labortechnik, Switzerland) with a 1.5-mm nozzle diameter and main spray chamber of 500 × 215 mm. The emulsion was then pumped to the dryer by a peristaltic pump with a flow rate of 5 cm 3 / min. The flow rate of the drying air was adjusted to 2.5 m 3 / min. and at a pressure of 0.06 MPa. The inlet and outlet air temperatures were 180-195 and 71-75 °C, respectively. The powdered microcapsules were stored for further characterization in air-tight desiccators. Morphological characterization of the produced nano-encapsulated cinnamon (NECO) and basil (NEBO) oils was analyzed using the Transmission electron microscope (TEM) (JEOL JSM6300 SEM, Tokyo, Japan), as described by Mohammed et al., (2017). In brief, the fresh samples of NECO and NEBO were placed individually onto a carbon-coated copper grid to form a thin liquid film, and then were negatively stained by adding one drop of uranyl acetate. The excess of staining solution was dragged using a filter paper, and the film was air-dried before observation. An Orius 1000 CCD camera (GATAN, Warrendale, PA, USA) was used for the image acquisition. The average diameter, zeta potential (ZP) and size distribution of the NECO and NEBO were measured using a particle size analyzer (Nano-ZS, Malvern Instruments Ltd., UK). For measuring zeta potential, each sample was sonicated for 30-60 min. before assessment, in reference to Abdel-Wahhab et al., (2018).

Collection of swab samples
About 115 wound swab samples were collected from patients admitted to the Naser Institute, Cairo, Egypt, from both sexes with ages that ranged from 14-60 years with complaints of discharge; pain, swelling, foul-smelling, delayed and non-healing wound infections. Sterile cotton swabs were used for samples collection by gently swabbing the interior surface of the infected area, and then the swabs were transported immediately to the microbiology laboratory for bacterial isolation.

Isolation and identification of the bacteria
Each swab sample was streaked individually onto the surface of 3 Petri plates of McConkey agar; blood agar and nutrient agar (NA) (Oxoid Ltd. Co, Nepean, ON, Canada), and then all plates were incubated at 37°C for 24 h. After incubation, the growing bacterial colonies were observed through the formation of macroscopic colonies. The isolated bacteria were purified, and then identified phenotypically using cell morphology and Gram staining. Biochemical identification of the isolates was carried out using several biochemical assays, according to Forbes et al., (1998);Federal Drug Administration. (2001). Furthermore, characterization of the bacterial isolates was confirmed using API 20 kits, as recommended by the manufacturer's instructions (Biomereux, France).

Antibacterial potential of the essential oils
Using MHA medium, about 20 ml of this medium was dispensed into sterile Petri plates, seeded with 100 µl of 10 8 cells/ ml of broth culture of each tested MDR bacteria, and then spread on the plate surface using a sterile glass spreader (El-Mahmood and Doughari, 2009). Filter paper discs (6 mm) were impregnated with different concentrations (10-50 µl) of 40 % encapsulated essential oils (NECO and NEBO), and then placed aseptically on the surface of the seeded plates. After incubation of the plates for 24 h at 37°C, the diameters of inhibition zones surrounding the discs were measured (mm) using a calibrated ruler. Three replicate plates were used for each tested bacterial strain, and the assay was repeated 3 times.

Statistical analysis
Three replicates plates were used for each tested concentration of the essential oils, and the assay was replicated 3 times. The significances of the tested essential oils based on the zones of inhibition were determined using analysis of variance (ANOVA) at the 5 % level. Means were compared with Duncan's multiple range test of SAS statistical analysis system.

Composition of the essential oils
Essential oils (EO) are complex aromatic substances derived from plants, which are composed mainly of complex mixture of polar and non-polar compounds including terpenes (Dhifi et al., 2016). According to El-Nekeety et al. (2021), GC-MS analysis of the basil essential oil used in the current study revealed the presence of 48 compounds. In accordance, other studies also revealed that Linalool was the major compound that exists in basil essential oil (Benedec et al., 2013;Amor et al., 2021). However, a previous study conducted by Olugbade et al., (2017) stated that the major components of basil oil were methyl eugenol followed by methyl chavicol, whereas another study of Ghasemi Pirbalouti et al., (2017) reported that methyl chavicol was the major compound followed by linalool. On the other hand, the GC-MS analysis of cinnamon essential oil used in this study was carried out by Mohammed et al., (2020), and notified the presence of 16 compounds. Similar results were previously obtained by Wang et al., (2009);Abdel-Wahhab et al., (2018), who reported that trans-cinnamaldehyde was the major compound present in cinnamon essential oil. Conversely, Raina et al., (2001) found that eugenol, linalool and pipertone were the major 3 components in ECO, and the recorded concentrations of these compounds were 76.6 %, 8.5 % and 3.3 %, respectively. The variation between these results may be attributed to several factors such as; the existence of numerous botanical varieties, cultivars and chemotypes (Filip, 2017), climatic factors (Milenkovic et al., 2019), and the part of the plant used during extraction of the oil (Kaul et al., 2003).

Characterization of the NECO and NEBO
The TEM analysis demonstrated that NEBO (Fig.  1A) and NECO (Fig. 1B) have a smooth rounded shape, whereas the size analyses revealed that the average particle size of NEBO was 120 nm, while the average size of NECO was 130 nm. The recorded ZP for NEBO and NECO were -4.73 and -17.85 mV, respectively. These current results expressed that the use of WP enhanced the droplet coalescence, in agreement with Goula and Adamopoulos, (2012). Moreover, the uniform size distribution of the oil droplets and their smooth round shape indicated the formation of a droplet wall material by WP (Xu et al., Novel Research in Microbiology Journal, 20212013Eratte et al., 2014). A previous study conducted by Roger et al., (2010) reported that the shape, surface property and size of the particles have critical roles in the uptake of nanosized particles by the cells, and added that the size between 50-300 nm has a favorable uptake than the other counterparts. Besides, Sadat et al., (2016) revealed that the size of NPs affects the distribution, clearance and the pharmacokinetics of these NPs. Meanwhile, ZP plays an important role in the distribution of the oil droplets and increases their stability (McClements and Rao, 2011). The negative charged ZP reported in this study for the NEBO and NECO might be attributed to the negative charge of the carboxylate group in WP, which is the only functional charge in the globulars of WP, in agreement with Eratte et al., (2014).

Identification of the isolated bacteria
In this study, a total of 115 pure bacterial isolates were recovered from the collected swab samples. Results of the phenotypic characterization and the characteristics obtained by API 20 kits indicated that these 115 isolates belong to 6 different genera of bacteria including; E. coli, Staphylococcus aureus, A. baumannii, P. aeruginosa, K. pneumonia and Proteus vulgaris ( Table 1). The frequency of these bacterial strains are arranged in descending order according to their prevalence as Staphylococcus aureus (30 isolates, 26.1 %) > P. aeruginosa (29 isolates, 25.2 %) > K. pneumoniae (27 isolates, 23.5 %) > A. baumannii (14 isolates, 12.2 %) > Proteus vulgaris (9 isolates, 7.8 %) > E. coli (6 isolates, 5.2 %), as shown in Table (2). These strains are common contaminants of wounds in agreement with the previous results reported by Otokunefo and Brown, (1990); Adebayor et al.,

Antibiotics susceptibility assay
The antibiotic resistance pattern of all the identified strains is given in Table (

A B
Novel Research in Microbiology Journal, 2021 However about 73.3 % and 76.7 % of Staphylococcus aureus strains were sensitive to meropenem and imipenem, respectively. Similarly, resistance of Staphylococcus aureus bacteria that was isolated from infected wounds and cases of bacteraemia was reported previously by Takeda et al., (2000). The mechanism of this resistance may be attributed to several genetic factors including; mutations, ticking of the cell wall, production of ßlactamase and modification of the receptor site(s) specific for antibiotics (Hiramatsu et al., 2014). The results presented in Table (3) also showed the antibiotic profile of P. aeruginosa strains, which expressed resistance to amikacin; ampicillin; cefotaxime; ceftazidime; ceftriaxone; ciprofloxacin; gentamicin; ofloxacin; tetracycline and tobramycin The resistance of these strains was reported previously by Harvey et al., (2010), who suggested that P. aeruginosa is able to make biofilms in the patients, and this bacterium could also be transmitted from the wounds to the blood circulation; leading to either gastrointestinal or urogenital infections as recorded in the AIDS patients. The antibiotic sensitivity profiles of K. pneumonia, A. baumannii, Proteus vulgaris and E. coli (Table 3) revealed that K. pneumoniae strains showed susceptibility to imipenem (7.4 %) and meropenem (11.1 %). On the other hand, about 100 % of A. baumannii strains can resist amikacin; amoxicillin, ampicillin and cefaclor; however, Proteus vulgaris strains were susceptible to imipenem and meropenem by 100 % and 88.9 %, respectively. Furthermore, all E. coli strains showed sensitivity to meropenem and imipenem. Although 50 % of these E. coli strains were susceptible to ceftazidime and ceftriaxone; however, 100 % of them expressed resistance to tetracycline. The remaining antibiotics showed variability in their effects against E. coli strains. The antibiotic resistance pattern of K. pneumoniae, Proteus vulgaris and E. coli was reported previously by several authors (Todar, 2007;Li et al., 2015;Fodor et al., 2020), and the mechanisms of this resistance may be probably attributed to genetic reasons or due to modifications of the cell source proteins, as reported by previous studies conducted by Enan, (2006a, b); Ouda et al., (2014); Abdel-Shafi et al., (2014). The antibiotic resistance of all the tested bacterial strains that were isolated from wounds confirmed the relationship between these bacteria, wound infections and wounds associated bacteremia.

Antibacterial potency of NEBO and NECO
In the present study, NEBO and NECO were applied as antibacterial agents. The data presented in Table (4), Fig. (2, 3) represent the antibacterial efficacy of various concentrations of NEBO and NECO (10 µl, 20 µl, 30 µl, 40 µl and 50 µl) against the currently identified six MDR bacterial strains. Results indicated NEBO and NECO have antibacterial efficacy against either all or some of the tested strains. NEBO showed a strong inhibitory effect against Staphylococcus aureus strains and a weak inhibitory effect on Proteus vulgaris and E. coli strains, but it did not show any inhibitory activity against P. aeruginosa, K. pneumonia and A. baumannii. However, NECO exhibited a strong antibacterial potency against Staphylococcus aureus, A. baumannii, Proteus vulgaris and E. coli; however, a good inhibitory effect was expressed against P. aeruginosa and K. pneumoniae.
Previous reports showed that NEBO exhibited significant antibacterial potential against both Gram (-) and Gram (+) bacteria, where the Gram (+) bacterial strains expressed more sensitivity to this oil (Suppakul et al., 2003;Hussain et al., 2008;Vanin et al., 2014;Dhifi et al., 2016;Amor et al., 2021). This effect may be attributed to the high contents of Linalool, which was reported to have a strong antibacterial activity against Gram (+) than Gram (-) bacterial strains (Ebrahim Sajjadi, 2006;Sokovic and Van Griensven, 2006). This compound may cause disruption of the bacterial cell membrane leading to the cell death.
Novel Research in Microbiology Journal, 2021 Amikacin (30 µg   The antibacterial efficacy of NECO may be due to its high content of cinnamaldehyde, which previously demonstrated moderate antibacterial potency; however, this effect was more pronounced against Staphylococcus aureus than E. coli (Raeisi et al., 2015;Alizadeh Behbahani et al., 2020). These results indicated that the Gram (+) bacteria are more antibiotic resistant than Gram (-), due to the difference in their membranes structures.
The encapsulation process protected the oils against any oxidation or evaporation (Suppakul et al., 2003), and increased their stability (Liang et al., 2012;Terjung et al., 2012). Moreover, encapsulation increased the bioavailability, and provided control release of the bioactive compounds in the oils (Majeed et al., 2015). Similar to the current results, Dev Kumar et al., (2020) reported that encapsulation of the basil essential oil increased its potential antimicrobial and antioxidant efficacies.
In the current study, another mechanism for enhancement of the antibacterial activity of the encapsulated oils may be attributed to the antimicrobial activity of WP used during the encapsulation process. Previous reports of Perez-Gago and Krochta, (2002) suggested that the antimicrobial films made by WP were excellent barriers, flavorless; transparent and flexible. For these properties, WP films were widely used to carry the antimicrobial agents targeted against Staphylococcus aureus and Listeria monocytogenes (Oussalah et al., 2004, Royo et al., 2010. The antimicrobial efficacy of WP is mainly due the presence of some minor proteins such as immunoglobulins (Igs); lysozyme (Lyz), lactoperoxidase (LP) and lactoferrin (Lf) (Yamauchi et al., 2006). Lf and Lyz work synergistically as antibacterials against Gram (+) bacteria, and inactivate Gram (-) bacteria such as Salmonella enteritidis (Uniacke-Lowe et al., 2010;Detha et al., 2018). Consequently, encapsulation of the essential oils using WP may be promising for enhancement of the antibacterial activities of EBO and ECO against the MDR Gram (+) and Gram (-) bacteria.

Conclusion
The current study revealed that 6 genera of bacteria including E. coli; Staphylococcus aureus, A. baumannii, P. aeruginosa, K. pneumonia, and Proteus vulgaris, were isolated from the wound samples collected from patients suffering from pus discharge; pain, swelling, foul-smelling and non-healing wound infections that are mainly of bacterial origin. Most of the isolated strains were resistant to amikacin; ampicillin, cefotaxime, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, ofloxacin, tetracycline and tobramycin. The nano-encapsulated essential oils of basil and cinnamon (NEBO and NECO) showed potential antibacterial activities against the MDR bacteria. The encapsulation process may be effective to enhance the antibacterial potentials of the essential oils, since it protects the components of the oils from oxidation. Therefore, these encapsulated oils may be promising agents for possible application in treatment of the MDR bacteria. Nanoencapsulation of basil essential oil alleviates the oxidative stress, genotoxicity and DNA damage in rats exposed to biosynthesized iron nanoparticles. Heliyon. 7: e07537. https://doi.org/10.1016/j.heliyon.2021.e07537