FT-IR spectroscopy
Fourier transform infra-red spectra for most bacteria have four distinguishable regions. Region I (3000-2800 cm-1) represents cell membrane fatty acids, with three noticeable peaks (2960, 2925, and 2860 cm-1)[11, 14]. Region II (1700-1500 cm-1) shows amide I (1650 cm-1) and amide II (1550 cm-1) bands of proteins and peptides[16]. Region III (1500-1200 cm-3) corresponds to fatty acids as well as proteins and phosphate-carrying molecules. Three major peaks at 1455 cm-1, 1400 cm-1 and 1240 cm-1 depict changes to lipids and proteins; carbohydrates and nucleic acids or phospholipids, respectively. Bands at 1080 cm-1 are also related to nucleic acids (11). Region IV (1200-900 cm-3) shows absorption bands typical of polysaccharides or carbohydrates of microbial cell walls with an absorption peak between 1100-950 cm-1[14, 16].
The spectra obtained for all the tested Bifidobacterium strains were similar to those previously described for other bacteria[11, 14, 23]. Strong absorptions were obtained for all four spectral regions (4000-850 cm-1) representing the main components of a cell. Control and GCE-treated Bifidobacterium samples showed discrepancies in spectra obtained, as discussed in detail for each tested strain below.
Bifidobacterium bifidum LMG 11041
Spectral features of control and GCE treated samples were different (Figure 1). There were shifts at all major peaks, 3285, 2930, 1655, 1550, 1452, 1400, 1238, 1078, and 913 cm-1 (Figure 1), indicating changes in the biochemical composition of cells due to exposure to GCE. Shifts at peaks 3285 cm-1 indicate changes to proteins and polysaccharides while shifts at peak 2930 cm-1 correspond to changes to lipids. In addition, there was an increase in spectral frequency at peak 2934 cm-1, which may indicate an increase in membrane fluidity as well as a conformation disorder of the acyl chains of the cell membrane phospholipids[13]. These results suggest a change in properties of cell membranes of treated cells. Minor shifts at peaks 1649 cm-1 and 1544 cm-1 (Amide I and II) were also observed, with an associated increase in intensity of spectral features of these two peaks for GCE-treated cells compared to the control. This could indicate an increase in polysaccharides, which serve as a mechanism of survival for cells to down regulate functions to save energy and protect themselves from stress[11]. The biggest shifts occurred at peaks 1238 cm-1 and 1079 cm-1, which represent phosphodiesters and nucleic acids, and carbohydrate regions in the cell wall, respectively. There were major decreases in spectral intensities of GCE treated cells for these peaks, which could indicate a reduction in viable counts, prevention of cell growth or cell death[11]. Allicin, the main active compound of garlic, can readily pass through cell membranes and affect lipid and fatty acid biosynthesis causing changes in viability of cells[10]. Changes in the nucleic acid region corresponds to published literature reports stating that allicin completely inhibits RNA synthesis and partially inhibits protein and DNA synthesis[6]. Furthermore, the spectra for control showed a clear peak at 913 cm-1, which disappeared after treatment of cells with GCE. This may indicate damage to the phospholipids in the cell wall. Similar observations were reported for Escherichia coli and Listeria monocytogenes due to their exposure to garlic[17].
Bifidobacterium longum LMG 13197
Similar to what was observed for B. bifidum LMG 11041, there were considerable shifts in the spectra at peaks 3291 cm-1 and 2928 cm-1 for GCE-treated cells (Figure 2). Compared to control samples, prominent changes were observed in region 3600-2800 cm-1, with decreased band area and intensities at peaks 3291 cm-1 and 2928 cm-1 (representing lipids), which could be related to a reduction in cell viability. A decrease in spectral band intensity is an indication of cell death. Similar results were previously reported for E. coli O157:H7 that were subjected to cold stress and low nutrient media[11]. Environmental stresses have been shown to induce changes in the cell membrane lipid composition[24]. Furthermore, allicin disrupts microbial lipid and fatty acid formation, thereby causing changes in viability of cells[10]. An increase in intensities of both amide bands for GCE-treated cells was also observed. These cells also showed a decrease in spectral intensities in regions 1490 cm-1 – 1260 cm-1 and 1074 cm-1, coupled with a reduction in band area at peak 1074 cm-1, changes usually associated with cell death or cessation of growth[11]. Shifts at peaks 1236 cm-1 and 1074 cm-1 suggest denaturation of the phosphodiester backbone of nucleic acids, thus damage to DNA and RNA. The peaks observed at 914 cm-1 and 871 cm-1 for controls were absent in GCE-treated cells, indicating changes to the structure of the bacterial envelope polysaccharides[23].
Bifidobacterium lactis Bb12
There were minimal discernible differences between spectral features for control and GCE treated B. lactis Bb12 samples at peaks 2930 cm-1 (lipids), 1648 cm-1 (Amide I), 1542 cm-1 (Amide II), 1453 cm-1 (proteins) and 1235 cm-1 (phosphodiesters) (Figure 3). The observed visual similarities between FTIR spectra for control and GCE treated B. lactis is attributed to its intrinsic resistance of detrimental factors, specifically its relative resilience to antibacterial effects of garlic, compared to other bifidobacteria[4]. However, a slight decrease in spectral intensity around the lipid regions (2929 cm-1) and a reduction in intensity around the phosphodiester region (1235 cm -1) for GCE-treated cells compared to controls were noticeable. As already mentioned, these are changes attributed to a decrease in cell viability[11]. According to Al-Qadiri et al.[15], a change in the spectral regions at peak 1235 cm -1 indicates nucleic acid denaturation. A major decrease in intensity for this strain was observed at peak 1073 cm-1, which is within a region corresponding to nucleic acids. Therefore, these observations once again indicated that GCE altered RNA and DNA, as was observed with the other Bifidobacterium strains tested in this study, although the change was less pronounced for this strain, and reported for other bacteria elsewhere, a damage that may eventually lead to cessation of growth or microbial death.
Principle component analysis of FTIR spectra
Principle component analysis has been extensively used for interpretation of infrared spectra in microbiology, medicine, agricultural and food sciences. It reduces a multidimensional data set to its most dominant features, removes random variation while maintaining the relevant variations between data points[16, 25]. It shows whether there are definite clusters in the data and describes similarities or differences from multivariate data sets[25]. In this study, PCA was performed concurrently for all four FTIR spectral regions described, as well as for the separate regions. Groupings of the spectra representing differences or similarities among the regions were then used to compare molecular compositions of control and GCE-treated bifidobacteria.
Figure 4 shows the PCA of the first derivative and multiplicative scatter correction of B. bifidum LMG 11041. Comparison of whole spectra patterns of the control and GCE-treated samples showed segregation between these two groups of samples (Figure 4E). However, upon closer inspection, distinct separation between clusters in region I (Figure 4A) and III (Figure 4C) were observed than in other regions, with differences better distinguished in region III than region I. There was no distinct clustering for in spectral regions II and IV (Figure 4B and D). These findings show that the most significant changes induced by GCE occurred in the region representing cell structure proteins and phosphodiesters associated with phospholipid bilayers, while other cellular constituents were less affected. This further explains our previous reports of unusual morphological changes for this strain[4].
Separation of spectra between B. longum LMG 13197 control and GCE-treated samples over all regions were observed (Figure 5). Major spectral differences were observed in regions I, II and III (Figure 5B-D) than in region IV. This indicated that for B. longum LMG 13197, polysaccharides of the cell wall were less affected than the other cellular components. Garlic clove extract caused more biochemical changes in this strain compared to B. bifidum LMG 11041.
Clear separations with distinct sample clusters were observed for control and GCE-treated B. lactis Bb12 throughout all the spectral regions (Figure 6). However, there were fewer differences between control and GCE-treated samples in regions III and IV as these clusters were closer to each other (Figure 6). Significant differences were observed within the lipid and protein regions, indicated by the more isolated clusters (Figure 6B-C). Damage was confined to the cell wall for this strain whereas for the more sensitive strains (B. bifidum LMG 11041 and B. longum LMG 13197), it extended to the nucleic acids.
In summary, PCA confirmed distinctive features of the FT-IR spectra among Bifidobacterium cells, indicated by clear segregations with distinct sample clusters in most if not all of the spectral regions between control and GCE-treated cells. In all the tested cells, exposure to GCE resulted in significant changes in lipids or fatty acids in the cell membrane, structural proteins and phosphodiesters associated with phospholipid bilayer. Noteworthy differences between control and GCE-treated cells were also observed in the amide groups of proteins, the nucleic acids as well as in polysaccharides of the cell walls for B. longum LMG 13197 and B. lactis Bb12.
Flow cytometric analysis
Double staining of Bifidobacterium cells with PI and SYTO9 was used to determine the effect of GCE on the cell membrane integrity, thereby giving an indication of whether the cell is viable, dead or damaged. Intact, undamaged cell membrane excludes the nucleic acid dye, PI, while damage permeabilizes it to this stain, which upon entry into the cells binds to nucleic acids, resulting in red fluorescence. Propidium iodide is therefore used as a marker for dead cells. SYTO9 stains all cells in a population, whether they are alive or dead[26]. It is however displaced by PI from the nucleic acids of damaged cells due to the higher affinity of the latter for DNA[21]. These stains have been used in studies on lactic acid bacteria to differentiate between healthy and damaged cells[19, 21, 27]. Figure 7 depicts the dot plots obtained for the different Bifidobacterium strains before and after treatment with GCE. Different quadrants were set to represent the following populations of cells: quadrant 1 (Q1): live SYTO9-stained cells, quadrant 2 (Q2): double-stained membrane damaged cells, quadrant 3 (Q3): dead PI-stained cells and quadrant 4 (Q4): unstained cells. There were noticeable differences between control and GCE treated cells for all the tested Bifidobacterium strains. The percentage of viable cells of B. bifidum LMG 11041 cells decreased by about 17% after GCE treatment while increases of 8.32, 0.1 and 8.08% were observed in Q2, Q3 and Q4, respectively (Figure 7, A1 and A2). Bifidobacterium longum LMG 13197 followed a similar trend to B. bifidum LMG 11041. The viable cell population decreased by 11% after GCE treatment, whereas number of damaged cells in Q2 increased by 9.3% (Figure 7, B1 and B2). The major difference between B. longum LMG 13197 and B. bifidum LMG 11041 was that an increase in the percentage of unstained B. bifidum cells, was less than that observed for B. longum by 7.4%. These results suggest that this strain was less susceptible to antibacterial effects of GCE. The viable population of B. lactis Bb12 was only 67% after GCE treatment compared to 99.7% for control sample. However, for this strain, the reduction in percentage of viable cells was accompanied by a substantial increase in percentage (24.77%) of unstained cells (Figure 7, C1 and C2), an increase higher than that observed for each of the other two strains by more than 15%. This result did not correspond to data obtained by FTIR, which indicated less damage to B. lactis Bb12. However, the percentage of cells having membrane damage was comparable to that obtained for the other two strains. Flow cytometric data results confirmed that all three Bifidobacterium strains tested were susceptible to GCE. An increase in percentage of cells in Q2 is evident of progressive cell damage and membrane deterioration. Similar flow cytometric data for bifidobacteria exposed to bile salt stress was obtained by Ben Amor et al.[28]. Ananta et al.[27] also found cell membrane damage using flow cytometry to analyze viability of Lactobacillus rhamnosus after spray drying.
A definite reason for the presence of unstained cells in Q4 is unknown, but it could be attributed to one or a combination of the following. Firstly, unstained cells could correspond to the cells that have undergone severe lysis and thus lost their nucleic acids, thereby rendering them unstainable[29]. These ‘ghost cells’ have been described by other researchers elsewhere[30, 31]. Previous studies have also reported that garlic causes cell lysis. Kim et al.[32] reported cell wall lysis in Listeria monocytogenes cells treated with garlic shoot juice. Allicin, the major active compound of garlic, has been reported have the ability to pass through phospholipid membranes, causing cell membrane damage and eventually cell lysis and death[8]. Secondly, these could be cells that clumped together or formed interlaced chains which, according to Hayouni et al.[33], may decrease staining accuracy.
Differences in shape and density of the populations’ light scatter patterns before and after exposure to GCE were also observed for all the tested Bifidobacterium strains. After GCE treatment the light scatter patterns became less diffuse and more concentrated, indicating a change in size, granularity and molecular content of the cells (Figure 7). Similar results were also observed in Candida albicans by Grannoum 1988, where the change in structure and integrity of the outer membrane was attributed to a decrease in lipid content of the membrane in the presence of garlic[32]. A different light scatter pattern obtained after exposure of cells to GCE could be due to a change in size or cellular structure and external morphology, whereby cells change from rod to coccoid shape. We have recently reported a change of bifidobacteria from rod to cocci shaped cells with cross- walls due to treatment with GCE[4]. Similar results were reported by Schenk et al.[34] for E. coli and L. innocua cells exposed to UV-C light. Young[35] also showed that typical rod-shaped Bifidobacterium spp. became coccoid under stress.