LIST OF TABLES
5.3. Results and discussion
5.3.3. Flow cytometric investigation
Traditional methods for assaying antibacterial activity are based on the visualization of bacterial growth in a qualitative manner. However, in the present study, along with the qualitative assays, the quantification of individual cells in the heterogeneous population has also been carried out by FC. Moreover, the sensitivity of flow cytometric analysis is significantly higher for the reason that the detectable change of the fluorescence histogram can directly correlate the bacterial cell membrane damage. In order to provide some insights into the mechanisms of action of all three extracts and isolated compounds on seven tested bacterial cells, the multiparametric FC technique was exploited.
Flow cytometric histograms and median fluorescence intensity (MFI) of PI-stained bacteria after treating with extracts are shown in Fig 5.1. Here, the negative controls (N-cell
populations in presence of respective solvents) showed the minimum relative fluorescence with respect to control cell populations (Fig. 5.1A-G). Conversely, the positive control (HK- heat killed bacterial population) showed significant increase in relative fluorescence in all tested bacteria (Fig. 5.1A-G) and confirms the major cell populations as damaged or dead.
The rightward shifting of fluorescence peak in the histogram was observed when the bacterial cells were treated with extracts as compared to control populations (Fig. 5.1). The histogram peak shifting occurs due to high PI fluorescence intensity from the treated and heat killed cells. Untreated cell population has its characteristic unaltered cell membrane whereas, treated and heat killed cell population has compromised cell membrane which allows PI to enter into the cell and stain the nucleic acids. The extents of damaged and dead cells were estimated on the basis of MFI with reference to histogram peak shifting. Responses among the seven tested bacteria varied with treatment of individual extract. Interestingly it was observed that shifting of fluorescence peak in the histograms (toward right) and MFI were maximum when the cells were treated with S-EtAc and S-Met extracts which indicates significant damage and depolarization of most of the tested bacterial cytoplasmic membrane (Fig. 5.1).
Fig. 5.1 Flow cytometric histograms of PI-stained seven tested bacteria at their respective MIC values for each extracts. Histogram analysis has been done by using FlowJo software (Tree Star, Stanford, USA) and plotted as PI fluorescence (FL2-H) against total cell counts.
(A)-(G) represents overlay histograms and median fluorescence intensity (MFI) of PI (FL2- H) for S. aureus (SA), B. cereus (BC), L. monocytogenes (LM), E. coli (EC), S. paratyphi A (SP), E. coli enterotoxic (EE), and Y. enterocolitica (YE), respectively. C untreated bacteria (control), N bacteria treated with ethanol (negative control), HK heat killed bacteria, S-Hex, S-EtAc and S-Met are bacteria treated with seed hexane extract, ethyl acetate extract and methanol extract, respectively. Significant increase in MFI and peak shifting was clearly observed in each case with respective treatments.
Similarly, FC analysis was carried out to assess the effect of compound I and II on bacterial cell membrane integrity. Flow cytometric histograms and median fluorescence intensity (MFI) of propidium iodide (PI) stained bacteria are shown in Fig. 5.2. Alike the FC analysis with extracts, the vehicle control (cells treated with ethanol) showed minimal changes in relative fluorescence intensity with respect to control cell populations (Fig. 5.2A-G). While, the positive control (heat killed bacteria) showed significant increase in relative fluorescence intensity for all the tested bacteria (Fig. 5.2A-G) and confirmed the cell damage or death.
Increase in fluorescence similar to those observed for the positive controls were also observed when the bacterial cells were treated with compounds I and II. MFI values indicated that treatment of bacteria with compounds I and II caused significant (Tukey’s test, p<0.001) increase in fluorescence intensity compared to the vehicle control for all seven bacterial strains, with compound II having a significantly more pronounced effect than compound I, especially for the Gram-negative bacterial strains (Tukey’s test, pE. coli = 0.001, pS. paratyphi
= 0.038, pE. coli enterotoxic
= 0.009, and pY. enterocolitica
<0.001).
Flow cytometric technique has become a promising tool in wide range of application, including antibacterial activity studies by detecting the change in bacterial membrane potential, permeability and even for the development of rapid antibacterial drug discovery in recent years (Durodie et al. 1995; Caron et al. 1998; Novo et al. 2000; Gnanadhas et al. 2013;
Scanlon et al. 2013; Stankovic et al. 2013). Previously, flow cytometric investigation on L.
monocytogenes and S. aureus revealed the antibacterial effect by various plant essential oils (Nguefack et al. 2004). The mode of bactericidal effect of essential oil was found similar to the present study which possibly due to the permeabilization of cytoplasmic membrane of bacteria.
Fig. 5.2 Flow cytometric histograms of PI-stained seven tested bacteria at their respective MIC values for each compound. Histogram analysis has been done by using FlowJo software (Tree Star, Stanford, USA). The figure represents overlay histograms and median fluorescence intensity (MFI) of PI (FL2-H) for (A) S. aureus (SA), (B) B. cereus (BC), (C) L.
monocytogenes (LM), (D) E. coli (EC), (E) S. paratyphi A (SP), (F) E. coli enterotoxic (EE), and (G) Y. enterocolitica (YE) respectively. C untreated bacteria (control), N bacteria treated with ethanol (negative control), HK heat killed bacteria. Significant increase in MFI and peak shifting was clearly observed in each case with respective treatments.