Typhoid fever, a potentially fatal illness is caused by S. typhi and S. paratyphi Gram-negative bacteria. Towards the end of last century, these bacterial strains developed multidrug resistance to most of the available first-line treatment drugs (e.g. chloramphenicol, ampicillin, etc.) in various parts of the world including Pakistan [44]. This resistance shifted the focus of medical science to more effective classes of antibiotics, quinolones and cephalosporins for the treatment of typhoid. But unfortunately, resistance emerged gradually against these drugs as well [7–9, 13, 45]. Among tested bacteria, all clinical isolates including three strains of Salmonella spp., E. coli and S. aureus have been identified as multidrug resistant strains [7, 37–40]. Essential oils are reported to posses, wide range of biological activities, especially these have been actively pursued as antimicrobial agents alone or in combination with other antibiotics. Notably, essential oils, when used in combination, have shown synergistic effects on the activity of antibiotics against which the microbes have already developed resistant. The essential oil of C. cyminum L decreased biofilm formation and enhanced the activity of the ciprofloxacin disk against K. pneumonia[46]. Similarly, this essential oil has improved the antimicrobial activity of nisin against food-borne pathogens [47].

The present study was conducted on essential oils of spices from Pakistani, which were extracted by hydro-distillation process. Different essential oils possessed significant (p <0.05) antimicrobial activity due to the presence of various bioactive compounds (Figure 1) [48]. Considering the MIC, C. cyminum essential oil was active against all tested bacterial species. It showed maximum activity against MDR S. typhi D1 Vi-positive and E. coli SS1 strains. This oil illustrated double activity (3.4 mg/ml) against S. typhi D1 Vi-positive (biofilm producing) strain than S. typhi G7 Vi-negative (biofilm non-producing) strain. It is generally believed that biofilm production is the characteristic which gives additional protection to the pathogenic microbes against host immune response and antibiotics [49, 50]. On the basis of these facts, Vi-positive strain can be assumed more resistant from antimicrobials than Vi-negative strain. However, in this study C. cyminum essential oil gave higher activity against Vi-positive strain. This may be due to the fact that the surface chemistry of these two types of Salmonella strains may be somewhat different. Moreover, essential oil from C. cyminum is known to inhibit or decrease biofilm formation and has enhanced the activity of the ciprofloxacin disk against Klebsiella pneumonia[46]. This oil may have halted the proper assembly of cell membrane, which ultimately inhibited Vi-positive bacterial growth. Nevertheless, application of essential oil from C. cyminum resulted in cell elongation, repression of capsule expression and inhibition of urease activity in biofilm producing K. pneumonia[51]. So, the use of C. cyminum essential oil alone or with combination of the existing antibiotics, could really contribute in developing the strategy to deal with the MDR biofilm forming microbial pathogens.

C. verum essential oil exhibited excellent response against all bacterial species with best MIC values (Table 1), which are in accordance with previous literature [52]. Particularly, it has strongly inhibited the growth of S. typhi G7 (Vi-negative) and P. fluorescens strains. In contrast to this, lower activity of C. cyminum essential oil against genus Pseudomonas has been observed [53]. In MIC assay, A. subulatum essential oil was found to be effective against all tested bacteria at lower concentrations especially against MDR E. coli SSI strain. No previous biological activities of cardamom essential oil were reported against the currently selected bacterial species except E. coli[54] whose MIC value did not correlate with our findings, which may be due to the fact that different species of cardamom used in previous study. Similarly, MIC assay revealed good level of biological activities against all of the tested microorganisms. Moreover, all tested bacteria showed resistance against amoxicillin in MIC assay except B. licheniformis exhibiting MIC value as 0.02 ± 0.05 mg/ml (Table 1, see footnote).

TLC analysis showed that cuminaldehyde was present as a main component in C. cyminum essential oil, as the colored zone showed R f value comparable with its reference standard. Presence of bands or zone of inhibition at same R f value in the extract showed antimicrobial activity against various microorganisms [55]. Bioautographic system expressed the antibacterial activity by zone of inhibition against bacterial species tested except P. fluorescens. These results correlate with the previous studies [53] where the C. cyminum essential oil has given good activity against various plants and mushroom disease causing bacterial pathogens, but this oil in general indicated lower inhibition activities against bacteria belonging to the genus Pseudomonas. However, no previous reports were found related to TLC-bioautography of C. cyminum essential oil against the other tested microorganism species. TLC-bioautography of C. verum essential oil confirmed the antibacterial activity of t-cinnamaldehyde against tested bacterial species except E. coli (Table 2) which were in agreement with the findings of Horvath et al. [42]. TLC analysis confirmed the presence of eucalyptol as major constituent of A. subulatum essential oil by comparing with reference standard as colored zone by calculating R f value. Bioautography showed that A. subulatum essential oil possessed antibacterial active agents by showing inhibition zone against all bacterial species except S. aureus and P. fluorescens. So far, no previous study is known related to TLC-bioautography of A. subulatum essential oil against the tested bacteria.

TLC-bioautography of S. aromaticum essential oil indicated the presence of bioactive agents by showing zone of inhibition against tested strains except P. fluorescens. These results were comparable to the previous findings [56]. TLC-bioautography of S. aromaticum essential oil against S. aureus revealed that lower concentration was effective in inhibiting tested bacteria which was more or less similar to the known literature [48]. Some of essential oils did not show inhibitory zones on TLC-bioautography against some bacterial strains, while they have showed activities in MIC assay in few cases. This might be due to the loading of less concentration of bioactive compound on TLC or evaporation of compounds and/or photo-oxidation [55].

Variation in the chemical profile of essential oils could influence their biological activities. Therefore, it was important to determine the chemical composition of essential oils to correlate with their antimicrobial activities [28]. Chemical profiling of essential oils were performed using GC/MS [15, 43]. The analysis of C. cyminum essential oil indicated the presence of eight volatile components (Figure 2 and Table 3), characterized mainly by monoterpene hydrocarbons (α-pinene, β-pinene, ρ-cymene and γ-terpinene) and oxygenated monoterpene (1, 8-cineole, cuminaldehyde, cuminyl alcohol and safranal). Previous reports on chemical composition of C. cyminum essential oil showed that it had oil yield of 5.4% with major compounds cuminaldehyde, 1, 8-cineole, ρ-cymene, γ-terpinene and β-pinene which are almost similar to our results [36, 57].

t-Cinnamaldehyde was identified as major component and eucalyptol as minor component in C. verum essential oil through GC/MS analysis (Table 3). These results were in accordance with previous reports [34] with some difference in concentration which could be due to seasonal variation, adaptive metabolism, parts of plant used, distillation process and other factors [58]. The GC-MS analysis of essential oil from A. subulatum (large cardamom) revealed oxygenated monoterpenoid eucalyptol as a major component and hydrocarbon monoterpene in traces (Table 3). While in literature volatile components of Amomum cannicarpum consisted of β-pinene, elemol and α-cadinol identified by GC/MS [35]. These results were found to be in contrast to our chemical profiling, which could be due to different sub-species comparison. The chemical composition of S. aromaticum essential oil showed the presence of oxygenated monoterpenes with eucalyptol as major component and eugenol in minor quantity. These results were not associated with previous studies [59], where chemical composition of S. aromaticum essential oil revealed eugenol, caryophyllyne and eugenyl acetate as major components. These variations in the chemical composition of essential oil might be due to ecological, climate and geographical conditions, time of harvesting and age of plant [60].