 revealed by the one-way ANOVA, there was a significant overall difference (P < 0.05) in the expression of each of the tested genes among the different groups. The  using Bonferroni's correction were performed to determine whether differences in expression of the tested genes between the exposed and control group were significant. These results were included in Fig 2. All genes, with the exception of only two genes [ycfR (BhsA) and evgA] were downregulated following exposure to all natural honeys under study (Fig 2). Although different degrees of downregulation were observed following exposure to the various honeys tested, all the a TEM (at 10,000 × magnification) and photographed (see Materials and Methods). The images represent E. coli cells: (A) of untreated control, (B) after treatment with artificial honey, (C) after treatment with citrus honey, (D) after treatment with clover honey, and (E) after treatment with marjoram honey. Scale bar = 500 nm (and is the same for A-E).
doi:10.1371/journal.pone.0150984.g001 coli ATCC 8739 to the tested honeys as determined by qPCR. Logphase E. coli ATCC 8739 cells were exposed for 3 h to sub inhibitory concentrations (0.3× MIC) of clover, citrus, marjoram, or artificial honey. Following cell harvest, total RNA was isolated, reverse-transcribed to cDNA, and the expression levels of eight target genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism were examined using qPCR (see Materials and Methods). Experiments were run with three technical replicates of each. Mean values of fold changes (± SD) are shown in relation to untreated (control) E. coli ATCC 8739 cells. Asterisks indicate statistically significant differences in the expression of each gene between treated samples and control, as analyzed using the one-way ANOVA with Bonferroni's correction for multiple testing (*p 0.05; **p 0.01; ***p 0.001; ns, no significant difference). downregulated genes showed less than six-fold change, except for the tnaA gene that was downregulated in the range of 12.5-16.2 fold (Fig 2).
In the case of ycfR (BhsA) gene, its expression was upregulated following exposure to the three tested natural honeys, while the upregulation of evgA was observed only in clover honeytreated cells, but not in citrus or marjoram honey-treated ones. The fold increase in expression of the ycfR (BhsA) gene was in the range of 2.2-4.19 fold, while evgA was upregulated by 1.09 fold following exposure to clover honey (Fig 2).
To evaluate the contribution of osmolarity and acidity on the level of expression of the target genes, this assay was also performed on cDNA synthesized from RNA extracted following exposure of the cells to an artificial honey solution. In the presence of artificial honey, downregulation in the expression of all the tested quorum-sensing genes, and upregulation in the expression of all those invovled in stress survival were observed with E. coli ATCC 8739 (Fig 2). In the case of biofilm-forming genes, exposure of E. coli ATCC 8739 cells to artificial honey caused a decreased expression of yjfO (bsmA), while it caused an increased expression of csgA, and ycfR (BhsA) (Fig 2).

Discussion
The ability of different types of honey to combat infections may be attributed to at least two complementary mechanisms. The first mechanism is attributed to their direct biocidal activity, owing to the presence of multiple factors that can damage susceptible organisms [80]. The second mechanism is mediated through their anti-virulence activity, by downregulating the expression of genes associated with virulence factor production, stress tolerance, and/or multicellular behaviors of the target organism (such as biofilm formation and quorum sensing) [80]. This latter mechanism will eventually weaken bacterial coordination, decrease their survival abilities, and interfere with their virulence mechanisms. In the current study, we investigated the antibacterial and the anti-virulence activities, in addition to the cellular changes, exerted by three Egyptian honeys from different floral sources (namely, citrus, clover, and marjoram) on E. coli ATCC 8739. To our knowledge, this is the first attempt to assess the impacts of Egyptian honey varieties on the tested organism at both structural and molecular levels. A summary of the obtained results is represented in Fig 3. It is well known that the total antibacterial activity of honey is attributed to multiple factors, including its high osmolarity, acidity, in addition to its content of hydrogen peroxide (H 2 O 2 ) and non-peroxide phytochemical components [81]. Therefore, the current study assessed these individual antibacterial factors and evaluated their relative contributions to the overall antibacterial activities of the honeys under study. To examine the effect of peroxide, the MIC values were determined in the presence and absence of catalase enzyme, which is known to break down H 2 O 2. The possible contribution of pH and acidities (free, lactonic and total) in solutions of the tested honeys were investigated. In addition, the probable contribution of osmolarity was assessed by including an artificial honey solution in the different assays performed.
In the absence of catalase, the MIC values of the tested honeys were 1.4-2.8 fold lower than that observed with the artificial honey. This may indicate that the phytochemical components exerted a more specific growth-inhibitory activity against E. coli than the osmotic effects of sugars. In this respect, several reports have shown the high antimicrobial activity of marjoram oil against both Gram-positive and -negative bacterial strains [82,83]. Similarly, a study conducted by Mishref and colleagues [84] has demonstrated that honey from bee colonies fed by medical herb extracts (such as marjoram) were superior in their antimicrobial activity than honey from control colonies. Our findings support and confirm those mentioned earlier, since among the honeys investigated, that derived from marjoram exhibited the highest total antibacterial activity against E. coli, as indicated by having the lowest MIC values in the absence of catalase (25% w/v). This type of honey also showed the highest levels of hydrogen peroxidedependent activity, as the removal of its H 2 O 2 (by catalase enzyme) caused the highest fold increase in MIC, resulting in an approximately three fold-shift.
In the current study, both citrus and clover honeys showed equal MIC values of 50% (w/v). These MICs were comparable, to some extent, with those obtained by El-Fadaly et al. [85], who reported equal antibacterial activities of Egyptian citrus and clover honeys against E. coli, with the MIC value of 35% (w/v). However, in contrast to our results, El-Kalyoubi, Khalaf [86] reported that Egyptian honey derived from citrus possessed higher antimicrobial activity against E. coli than that derived from clover, while the opposite order of activity was observed by Elbanna, Attalla [31]. These conflicting results may be attributed to the differences in geographical and seasonal sources, as well as harvesting, processing, and storage conditions of the honeys tested.
Turning to evaluation of acidity, the three tested honeys were acidic in nature, with pH values ranging from 3.98 to 4.33, which are likely to be low enough to inhibit the growth of many microorganisms [81]. The obtained values were in line with those of previous studies that reported a pH ranging from 3.8 to 5.2 among different Egyptian clover and citrus honeys [31,55,86]. Interestingly, although the total and free acidity values in marjoram honey were both less than the corresponding values of citrus or clover honeys, the antibacterial activity of the former was higher than the two latter. This observation is consistent with that of Badawy, Shafii [55], who found no correlation between free acidity of honey and its antibacterial activity against tested E. coli. The current TEM analysis were performed on mid-exponential phase organisms because they are known to be generally more susceptible to antibacterial agents than those in the stationary phase [20]. In the current study, the TEM micrographs demonstrated the antibacterial effects of the tested honeys on E. coli ultrastructure, with the appearance of ghost cells with invisible nucleoid, cells with increased vacuoles, and/or with irregular shrunken cytoplasm, in addition to cells with relatively less electron-dense material compared to untreated controls. Several factors in the tested honeys, operating singly or simultaneously, may be involved in these alterations in the bacterial cells. The first factor may be the oxidative stress caused by H 2 O 2 production, which is supported by the finding that the antimicrobial activities of the tested honeys are strongly peroxide-dependent. The second factor may be the acidity of honey, because this low pH is likely to: (i) inhibit the metabolism of most enzymes, (ii) alter the fatty acids responsible for cell wall synthesis, and (iii) cause protein denaturation [67]. Since inactivation of metabolic enzymes can lead to accumulation of cells with decreased size, this may explain why most of the honey-treated cells were considerably smaller than the control ones. With prolonged exposure periods (more than the 3 h applied in the current structural studies), lysis of the bacterial cells, due to alteration in membrane integrity, may eventually occur. It is noteworthy that, while the osmotic effect of sugars could be one of the involved factors, it appeared to play a minor role, if any, to the ultrastructural changes reported above. This notion is supported by the absence of obvious morphological or structural abnormalities in the artificial honey-treated cells in this study.
A number of