Four gram negative and one gram positive bacterial strains namely Serratia liquefaciens, Serratia fonticola, Escherichia coli, Citrobacter freundii and Staphylococcus aureus respectively were selected. These tested microorganisms were recovered from street foods vended in the city of Karachi [11].

Four ethanolic lipid fractions namely Nigella sativa (black cumin), Coriander sativum (coriander), Foeniculum vulgare (fennel), and Laurus nobilis (bay leaf) were selected to assess their antimicrobial potential against selected strains [12]. These lipid fractions were collected from Food Science and Technology, University of Karachi by solvent extraction procedure [13].

Volumetric measurement was performed to find out the direct correlation between LFs and antibacterial activity against selected food borne bacteria. 18 hour’s old cultures were standardized in physiological saline and matched with 0.5 concentration of McFarland index. 100 µL bacterial inoculum was added and make thin and uniform bacterial lawn on Mueller Hinton agar plates with the help of sterile cotton swabs. Five sterilized filter paper disc of 0.6 cm soaked in 0.5, 1, 2, 5, and 7.5 µL concentrations of LFs were positioned onto MHA plates and incubated at 37˚C for 18 - 24 hours. Next day observed zone of inhibition around each disc [14] [15] [16].

This assay was used to examine the antibacterial activity of four ethanolic LFs in their concentrated form [17] [18]. The Mueller Hinton agar plates were formerly inoculated with test organisms with 0.5 McFarland concentration. 10 µL drop of LFs was placed on MHA plates and left untouched for proper dispersion at ambient temperature. After a while, plates were incubated at 37˚C ± 1˚C for 24 hours. Zone of inhibition around each drop was measured in millimeters.

Antibacterial activity of ethanolic LFs at different concentrations (1000 µg/mL, 500 µg/mL, and 250 µg/mL) prepared in DMSO was determined by agar well diffusion method as described by Martins et al., 2013 and Mehboob et al., 2020. To analyze the combined effects of ethanolic LFs were also evaluated by this method [2] [19].

To determine the minimal inhibitory concentrations of ethanolic lipid fractions in liquid media, broth dilution method was performed as described Weerakkody et al., 2010 at selected concentrations e.g. 1000, 500, 250, 125, 62.5, and 15.625 µg/mL prepared in DMSO Antibacterial activity of ethanolic lipid fractions based on the occurrence and absence of bacterial growth in the form of turbidity [2] [20] [21].

To measure the intracellular contents released from bacterial cell, loss of absorbance was estimated at 260 nm [22]. Working bacterial culture was centrifuged at 13,000 rpm for 15 minutes. 3x washing was done and cells were transferred to phosphate buffer saline. The concentrations of LFs used were same as broth dilution method and supplemented in bacterial cell suspension. Tested and control tubes were incubated for 2 hours in shaking incubator (CBM-Scientific.ES.20) at 150 rpm with incubation temperature 35˚C ± 2˚C. 2 hours later, 2 mL samples were filter sterilized by 0.2 µm pore sized filter membrane (Sigma Aldrich). The absorbance of the filtrate was measured at 260 nm via UV-V spectrophotometer Shimadzu, Japan 1240 Mini.

Levofloxacin (5 µg), streptomycin (10 µg), gentamicin (10 µg), amoxicillin (10 µg), tetracycline (30 µg) chloramphenicol (30 µg), and oxacillin (1 µg). The following antibiotics were purchased from Thermo Fischer Scientific Oxoid Ltd.

Scanning electron microscopy was performed with JEOL from Japan (model# JSM-6380) located at Centralized Science Laboratories, University of Karachi. Fresh culture of selected strains of Escherichia coli and Staphylococcus aureus (18 hrs. old) were standardized with 0.5 McFarland index. 10 µl of each selected bacterial suspension was inoculated into the micro centrifuge tube containing Mueller Hinton broth and MIC of LFs and incubated at 37˚C for 18 hours. Control tubes having bacterial suspension were also run alongside. After overnight incubation, 10 µL crystal violet was added in microcentrifuge tubes and let it stand for 1 minute. The tubes were centrifuged at 11,000 rpm for 10 minutes. After 3 washes with ethanol at 70%, 80%, and 90%, the specimens (bacterial cells) were coated up to 300 A˚ with gold using smart coater with an ion sputtering device (model JFC-1500) and energy dispersive X-ray spectroscopy detector (model EX-54175JMU; JEOL, Tokyo, Japan). Finally, morphological alterations in the bacterial cell before and after treatment were observed under scanning electron microscope.

According to the method proposed by Moussaoui & Alaoui, 2016 & Toroglu, 2007 combined effects of selected ethanolic LFs and antibiotics were examined through disc diffusion method [23] [24]. 10 µL drop of undiluted LFs were soaked on promptly accessible antibiotics disc, then plates were permitted to dry for proper diffusion, incubated at 37˚C ± 1˚C for 24 hours to find out the zone of clearance around each circle. The gained results were differentiated and tabulated between antibiotics + LFs and antibiotics alone by similar strategy as disk diffusion method.

For statistical evaluation, post hoc Duncan test was applied to find out the significant difference at p < 0.05 between control and tested sets of ethanolic LFs. All the tests were performed 3 times and results were presented as Mean ± SD by using SPSS software (version 24, SPSS Inc., USA).

All the four selected ethanolic LFs demonstrated no antibacterial activity at 0.5, 1, 2, 5, and 7.5 µl against all the tested bacterial strains.

Antimicrobial strength of concentrated ethanolic LFs was clearly explained with the aid of drop agar diffusion method. LF of FE exhibited significant antibacterial activity against all the tested bacteria and produced highest zone of inhibition ranges in between (11 ± 0 to 29.66 ± 0.57) mm among all the selected lipid fractions. Followed by LF of CE gave good competition and secured second position producing inhibition zone between (9 ± 0 to 24.33 ± 1.15) mm. While moderate antibacterial activity showed by LF of LE against all the tested bacterial isolates. However, LF of NE failed to retard the multiplication of S. fonticola, and C. freundii but demonstrated average antibacterial activity against E. coli, S. liquefaciens, and S. aureus with inhibition zone (9.93 ± 0.11) mm, (8.25 ± 0.4) mm, and (15 ± 0) mm respectively.

Antibacterial strength was also determined by agar well diffusion method (Table 1). All four ethanolic lipid fractions failed to inhibit the growth of S. fonticola and S. liquefaciens at all the chosen concentrations. While all of the lipid fractions showed antimicrobial potential against S. aureus and C. freundii. The lowest MIC of LF of NE and LE against S. aureus and C. freundii were 250 µg/mL. Correspondingly, the growth of S. aureus was inhibited by the LF of FE and CE at 1000 µg/mL. The MIC values of FE and CE were 500 µg/mL against C. freundii as shown in Table 2.

^{aN/D No detection of antibacterial activity. Values are means of triplicates ± SD.}

^{aN/D = No detection of antibacterial activity.}

In comparison to the well diffusion results, ethanolic lipid fractions demonstrated and produced enhanced antibacterial potential was detected in broth dilution method (Table 3). The highest and lowest MIC values of LF of FE were 1000 µg/mL for S. fonticola and 125 µg/mL for S. aureus, E. coli, and S. liquefaciens respectively. The least MIC value of LF of LE detected at 62.5 µg/mL against all the tested bacterial strains except C. freundii. Likewise, 125 µg/mL MIC value of LF of NE was noted against E. coli, S. liquefaciens, S. aureus, and C. freundii but for S. fonticola, 125 µg/mL MIC was observed. Furthermore, the MIC values of S. liquefaciens and S. fonticola were 500 µg/mL and 125 µg/mL against S. aureus and C. freundii while no activity was detected against E. coli.

Antimicrobial activity of lipid fractions was significant in broth medium therefore, antimicrobial potential was evaluated by estimating loss of absorbance at 260 nm. All the chosen LFs demonstrated significant difference between the tested and control group. The values were noted after 2 hours of incubation period. All four LFs specifically NE and FE at all the selected concentrations exhibited tremendous activity against S. aureus. The highly effected LFs were NE and CE against E. coli at 500 µg/mL. Similarly, highest destruction of bacterial cells of S. liquefaciens noted at 1000 µg/mL of CE. In case of S. fonticola, LF of NE at 250 µg/mL, LE at 500 µg/mL, FE at 1000 µg/mL and 500 µg/mL produced irreversible destruction to the bacterial cells. While LFs of NE, CE, and FE were effective against C. freundii at 1000 µg/mL, and 500 µg/mL respectively (Table 4).

Combined effects of standard antibiotics and ethanolic LFs were evaluated [25]. All the selected ethanolic LFs produced antagonistic effect against the tested food isolates when combined with selected antibiotics (Tables 5-8). Only LF of L. nobilis (LE) showed synergistic effect when combined with oxacillin and amoxicillin against C. freundii in Table 7. Also, synergistic effect was seen against S. liquefaciens when combination of LF of N. sativa (NE) and levofloxacin applied (Table 8).

Selected LFs combined to determine the antimicrobial strength against five tested food isolates. Noticeable antibacterial activity was not found against any bacterial strains at selected concentrations as demonstrated in Table 9. However, all the possible combination of ethanolic LFs proved significant activity against S. aureus with zone of inhibition ranges between (10 ± 0 to 15 ± 0) mm. Among them the most noteworthy and the minimal inhibition zone produced with the blend of N + L and F + L respectively.

Significant morphological and physiological changes appeared on bacterial cells after treated with ethanolic LFs. Cell wall became disrupted and it will lead to enhance the permeability of cell membrane. Due to increase in permeability, phenomena of cytolysis were seen and cell became swollen up and burst and all the internal contents released outside the cell (Figure 1).