A. baumannii has been described as AMR bacterium, and in this study, the isolates and type strain investigated for antimicrobial susceptibility to antibiotics demonstrated a range of susceptibilities to the different antibiotic groups tested (Table. 1). In 4/5 A. baumannii tested in this study, antibiotic resistance was identified and we observed varying resistance characteristics based on the criteria defined by Magiorakos et al. (2012). This included MDR in the ATCC 17978, PDR in the clinical isolate SM 37212 and possibly XDR in the ATCC BAA-1710™ isolate; however, further screening will confirm if this is a MDR or an XDR isolate. Following antibiotic screening, the isolates were exposed to lemongrass EO and major component (citral—approximately 90 % determined by GC-MS; Table 3) and found to be susceptible, including the isolates identified as MDR.
We observed that the human isolate ATCC BAA-1709™ was most sensitive to the antibiotics tested in this study and subsequently the most sensitive to the effects of both lemongrass EO and citral. Although it is difficult to make direct comparisons between the activity of the antibiotics and the EO/citral, the results suggest some similarity in level of activity between the different compounds. Other studies that have investigated activity of EOs and antibiotics have predominantly focused on the synergistic action (Rosato et al. 2007; Duarte et al. 2012; Aleksic et al. 2014
) rather than any comparisons between both compound types. However, there is a need for further research into how the effects of lemongrass EO and/or citral relate to the effect of antibiotics on drug sensitive and resistant bacteria. The MIC and MBC results suggest that the effect of citral is greater than that of whole lemongrass EO. This was observed previously in Adukwu et al. (2012) where the inhibitory and bactericidal concentrations of citral were also lower against different isolates of S.aureus further indicating that pure citral is more potent than whole lemongrass EO against different bacteria species.
Following determination of antimicrobial activity, we analysed the effect of the EO/citral on dermal fibroblasts.
In the study by Kpoviessi et al. (2014), the authors found that only C. citratus demonstrated a moderately toxic activity against the W138 cells (IC50 = 39.77 μg/ml) whilst the other Cymbopogon species had low cytotoxicity against same fibroblast cells and suggested the need for further toxicity studies. The authors also found that citral, approximately 75 % from GC-MS analysis in their study, also demonstrated a moderately toxic activity (IC50 = 39.48 μg/ml).
In our study, following exposure of the dermal fibroblast cells to lemongrass EO at the concentration 0.125 % (v/v), cell viability was reduced to approximately 60 %, and at 0.25 (v/v), cell viability was reduced to approximately 25 %. At 0.125 % (v/v), the effect of citral on cell viability was approximately 15–16 % greater than the activity of whole lemongrass EO on the fibroblast cells, and at 0.25 % (v/v), there was total loss in cell viability at the 1-h exposure time. There was no loss in cell viability at ≤0.03 % for lemongrass EO although following exposure to citral at the same concentration, there was approximately a 15 % loss in cell viability. Overall, comparing the effect of both whole lemongrass EO and citral the observation is that the difference in cytotoxic activity at the concentrations tested is approximately 20 % thus, suggesting that as the concentration is increased, the cytotoxicity of citral increases in favour of citral.
This is not the first time that the cytotoxic effects of EO components has been observed to be greater than the whole oil. In Hammer et al. (2006), the components of TTO were shown to be more cytotoxic against human cells lines in comparison to whole TTO. A similar response was found in the study by Prashar et al. (2004) which showed that both linalool and linalyl acetate (major components in lavender accounted for approximately 85 % of the oil) were more cytotoxic than the whole EO. In contrast, Hayes and Markovic (2002) showed similar cytotoxic action (similar IC50 results) and no significant difference when citral was compared with the Australian lemon myrtle EO. This is similar to the findings in our study as there was no significant difference between the IC50 of whole lemongrass EO and citral (P = 0.8254) and although the oils are different, the concentration of citral in our study (>89 % v/v) from GC-MS analysis (Table 3) was similar to the citral content in the Hayes and Markovic study (>92 % v/v).
Recommendations by the Nomenclature Committee on Cell Death (NCCD) are that at least two distinct methods of assessments should be used for cell death analysis (Kroemer et al. 2009), and in a follow-up paper by Galluzzi et al. (2009) on guidelines for use and interpretation of cell death assays, using methods such as enzymatic assays which involve the incorporation of exclusive dyes and long-term survival assays could be fundamental in answering the questions surrounding cell death as described by the NCCD. In this study, we used the XTT assay, now a common tool used in cell viability assays (Berridge et al. 2005) for determining the effect of the test compounds on cell survival and the live cell imaging for morphological examinations of the treated HDF.
Morphological examinations of the treated HDF showed that both lemongrass EO (1 % v/v) and citral (0.25 % v/v) caused damage to the fibroblasts within the first few minutes and total cell death within the 120-min exposure time at the tested concentrations. Close examination of the fibroblasts treated by lemongrass EO showed possible cell damage with features such as rounding up of the cells, cell shrinkage and retraction of the pseudopods suggesting damage as a result of apoptosis. The citral-treated fibroblasts showed an increased amount of blebbing (spherical membrane protrusions), cell retraction and shrinkage suggesting apoptosis as well as necrotic features on the damaged fibroblasts (Leverrier and Ridley 2001; Charras and Paluch 2008). The morphological changes and damage observed at the tested concentrations support the cell viability results which suggest lack of metabolic activity linked to the inhibition of cell proliferation thus cell damage and cell death features. To support the morphological observations, future studies would benefit from assessments to determine apoptotic and/or necrotic activity such as caspase activation and nuclear fragmentation assays as recommended by the NCCD (Kroemer et al. 2009) Also, further tests on cytotoxicity using this species of lemongrass in vitro and in vivo is needed on different cell lines before deciding whether or not to use this EO for management of human topical infections.
In summary, we have shown that lemongrass EO and citral were effective at inhibiting and killing MDR A. baumannii. This enhances the potential of the lemongrass EOs as a possible alternative to antibiotics owing to its ability to effectively inhibit and kill both MDR Gram-positive and Gram-negative bacteria. Our study also demonstrate that at concentrations shown to be bactericidal against Gram-negative drug-resistant A. baumannii, whole lemongrass EO and citral act against dermal fibroblast cells in vitro. However, at concentrations where citral and whole lemongrass EO were previously shown to be inhibitory and bactericidal against drug-resistant MRSA and MSSA (Adukwu 2013, PhD thesis), cell viability of the HDF cells remain high (≥70 %) which is a positive finding as it offers the potential for of these use in managing contamination in both clinical and non-clinical settings where drug-resistant staphylococcal infections remains a major problem globally.