Community associated or hospital-acquired infectious diseases resulting from multidrug-resistant pathogens are considered leading cause of morbidity and mortality, especially in developing countries (Laxminarayan et al., 2013). The injudicious use of antibiotics is the single most important factor leading to antibiotic resistance around the world (Centres for Disease Control, and Prevention [CDC], 2013). Center for Disease Control (CDC) has endorsed the need for development of new antibiotics. However, the development of new antibiotics is expensive and time consuming. Meanwhile the genetic plasticity in pathogens results in development of resistance at a rapid rate (Centres for Disease Control, and Prevention [CDC], 2013; Lee et al., 2013). To tackle this issue there is a dire need to search for safe alternative therapies (Jamil et al., 2015).

Natural products have garnered the attention of many researchers in recent years as complementary and alternative antimicrobial agents (Jeon et al., 2011; Calo et al., 2015). Essential oils (EOs) are natural plant products comprised of complex mixtures of biologically active substances and offer potential novel template molecules and bioactive compounds (Maida et al., 2014). EOs are composed of volatile secondary metabolites having antibacterial, antifungal, anti-inflammatory, antioxidant, anticancer, and antiviral activities (Espina et al., 2011; Hossain et al., 2014; Aumeeruddy-Elalfi et al., 2015; Sharifi-Rad et al., 2015). The efficiency of the EOs depends on its chemical composition, genotypes, environmental, and agronomic conditions (Mohamed et al., 2014). Recent studies have highlighted the antimicrobial potential of EOs with few reports against multidrug-resistant bacteria (Kon and Rai, 2012; Coelho and Pereira, 2013; Nazzaro et al., 2013). Furthermore, the use of EOs as food preservatives has also been described by many to control food pathogens (Djenane et al., 2013; Perricone et al., 2015). In the pharmaceutical field, EOs have been included in the composition of many dosage forms to impart flavor and natural preservation (Mohamed et al., 2014).

Despite the excellent antimicrobial activity of EOs against pathogenic microorganisms, their utilization is very limited due to low water solubility and less stability to environmental factors including heat, moisture, and oxygen. To improve water dispersion and protect EOs from degradation, nano-sized formulations emerge as a viable solution (São Pedro et al., 2009; Donsì et al., 2011; Acevedo-Fani et al., 2015; El Asbahani et al., 2015). Moreover, nano-encapsulation improves the antibacterial activity of several antibiotics (El Asbahani et al., 2015; Jamil et al., 2016).

Chitosan (CS) has proven to be a cost effective carrier for many pharmaceutical agents (Piras et al., 2015). Major characteristics that make it an ideal carrier system are its biodegradability, biocompatibility, availability, safety, cationic charge and innate antimicrobial potential. The present work has been done to investigate the microscopic, physico-chemical and cytotoxic attributes of cardamom EOs functionalized engineered CS nanoparticles (CSNPs). Not yet reported cardamom EO potential at nano-scale to treat multidrug-resistant pathogens was also explored which may provide a bio-based alternative to overcome existing therapeutic challenges posed by multidrug-resistant pathogens.

Cardamom, Lemon, rose, peppermint, eucalyptus, and orange EOs were purchased from local market. CS medium molecular weight was purchased from Sigma–Aldrich (Product number 448877, 75–85% deacetylation, 200–800 cP viscosity of 1% w/v in 1% v/v acetic acid). Pentasodium tripolyphospate (TPP) and glacial acetic acid were also procured from Sigma–Aldrich. All antibiotic disks were obtained from Oxoid.

Essential oils of plant origin are used both domestically and industrially. According to an estimate more than 3000 EOs have been produced by different plants. However, almost 300 are important from the commercial point of view. EO market all over the world is estimated to be 700 million US$ (Raut and Karuppayil, 2014).

In the present study we encapsulated EOs in CSNPs and then their antimicrobial potential on multidrug-resistant S. aureus and E. coli was determined. The S. aureus was confirmed to be MRSA because it was exhibiting resistance against cefoxitin disk. The proportion of S. aureus isolates that are resistant to methicillin is increasing rapidly. Previously, it was considered as nosocomial pathogen. However, infections caused by oxacillin (methicillin)-resistant S. aureus (MRSA) are a problem not only in health care institutions but also in community as well (Klevens et al., 2006). E. coli used in the study was ESBL positive. The prevalence of (ESBL) positive E. coli is also reported to be very high. According to a study it was estimated that almost 70–90% of E. coli population is ESBL positive (Kumarasamy et al., 2010). ESBL positive pathogens are considered to be resistant to whole cephalosporin range and the only safe treatment for curing ESBL positive pathogens is carbapenem and now the pathogens have developed resistance against carbapenem as well. So the clinicians are left with no safe antibiotics and there is a dire need to search for new antibiotics against which microbes cannot develop resistance rapidly.

Elettaria cardamomum (cardamom, family; Scitaminaceae) is a perennial herb, indigenous to India, Pakistan, Burma, and Sri Lanka (Khan et al., 2011). In addition to its wide use for culinary purpose, cardamom has folkloric repute as carminative, stomachic, diuretic, antibacterial, antiviral, antifungal and is considered useful in treatment of constipation, colic, diarrhoea, dyspepsia, vomiting, headache, epilepsy, and cardiovascular diseases (Gilani et al., 2008). Besides cardamom oil, the antimicrobial properties of several other EOs against a number of pathogenic microorganisms involved in foodborne illness have been demonstrated in previous investigations (Roby et al., 2013; Zhang et al., 2016).

Previous studies have reported MIC of cardamom EO at 0.4% against S. aureus ATCC 6538 and 0.8% against E. coli ATCC 8739 (Gochev et al., 2012). However, in the present study we tested the EOs against multi drug resistant pathogens that is why higher MIC values were achieved as compare to previous reports.

Numerous studies have reported an enhancement of the physical and antimicrobial properties of EOs-loaded nano-emulsions as compared to their conventional emulsions (Buranasuksombat et al., 2011; Liang et al., 2012; Salvia-Trujillo et al., 2014; Severino et al., 2015). The encapsulation of eugenol and carvacrol into nanometric surfactant micelles also resulted in enhanced antimicrobial activity. However, not yet reported previously, cardamom oil was encapsulated in CSNPs to protect it and to achieve antimicrobial activity at low dose below MIC value against MDR pathogens.

Hosseini et al. (2013) reported oregano loaded CSNPs by incorporating Tween, however, in the present study, cardamom oil-loaded CSNPs were prepared directly by ionic gelation method after mixing EO with TPP. It was observed after SEM (Figure 1) and AFM (Figure 2) analysis that NPs were in the range of 50–100 nm and they were uniformly distributed. CS is a very popular material as nano-carrier and has been widely studied by numerous authors. However, varying sizes in the range of 40–500 nm have been reported previously (Abreu et al., 2012; Hosseini et al., 2013).

While studying the interaction of cardamom oil with CS scaffolds, the materials were subjected to FTIR analysis. CS powder shows characteristic peaks that were in accordance to previously reported spectra (Khan et al., 2002; Vitali et al., 2006). The results of FTIR indicated that cardamom oil might be encapsulated into the CSNPs. Evidence of EOs and CS interaction was in good agreement with previous literature (Abreu et al., 2012). The CS and EOs-loaded CSNPs were subjected to stability testing by means of zeta potential analysis. Zeta potential studies predicted long term stability as the zeta potential values were more than + 60 mV in both empty and cardamom oil loaded CSNPs (Figure 4). Results of this study were in good accordance to the previous data (Calvo et al., 1997; Raja et al., 2013; Chang et al., 2015). A charge of more than + 30 mV is consider as an indicator of stability as the mutual repulsive forces would be more as compare to van der walls attractive forces between the NPs. In addition to stability the positive charge on both empty and loaded CSNPs indicated good interaction with pathogens as well. Bacterial cell wall carries negative charge and our developed NPs are carrying positive charge so there is a prediction of very strong interaction.

Our results of cytotoxicity were also found to be in accordance with the results of De Campos, where it was observed that both HCEC and HepG2 cell lines after CSNPs treatment yielded 100% cells viability (De Campos et al., 2004). Hemolysis or destruction of red blood cells is a very significant aspect to be considered for in vivo applications of all pharmaceutical agents as it can lead to anemia, jaundice and other pathological conditions. CSNPs were found to be absolutely non-hemolytic. Our results were found to be in good accordance to the findings of Zhou et al. (2015).

The antimicrobial potential of medicinal plants is well documented. Donsì investigated the nano-encapsulation of EO and has reported enhanced antimicrobial effect against multiple pathogens causing food spoilage (Donsì et al., 2011; Aumeeruddy-Elalfi et al., 2015). Moreover, Liang also reported enhanced and prolonged antimicrobial activity of EOs after nano-encapsulation (Liang et al., 2012). However, this is the first report on nano-encapsulation of cardamom oil in CSNPs. According to our findings cardamom oil loaded CSNPs were highly effective in combating both ESBL-producing E. coli and MRSA. Whereas, cardamom oil alone failed to eradicate them. However, the CSNP inhibited the growth of pathogens till first 48 h after that there was a rapid increase in optical density of both pathogens.

The positive charge due to the presence of –NH⁺³ have been mentioned as important factors that influence the antibacterial activities of CS (Chang et al., 2015). The strong adhesive properties of CS with bacteria are due to these opposite charges. However, to maintain the antimicrobial potential of CS, it must be incorporated with some antimicrobial agents and naturally occurring antimicrobials are the best choice for this purpose. It serves dual purpose, protecting the EOs from degradation for prolonged antimicrobial efficacy and sustaining the innate inhibitory effect of CS to inhibit resistant superbugs.

Cardamom EO-loaded CS nano-capsules were observed to be highly effective in controlling multi drug resistant E. coli and MRSA in vitro without showing any signs of toxicity to human cells.

BJ performed all research work and compiled data and wrote manuscript. RA performed cytotoxicity analysis and helped in manuscript write up. SA helped in collecting and identifying pathogens. MI helped for FTIR analysis. SK helped in studying growth kinetics studies on ELISA multi plate reader. AI helped in doing zeta potential analysis. SJ provided technical assistance in experimentation and write up. HB also provided technical assistance in experimentation and manuscript write up and MI^∗ provided technical assistance in experimentation and compilation of data and manuscript write up.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.