Table 1.
Primers used for the quantitative real-time PCR analysis in this study.
Table 2.
Acidity of the tested honeys (expressed as the mean of triplicate samples ± standard deviation).
Table 3.
Susceptibility of E. coli ATCC 8739 to the tested honeys.
MICs of citrus, clover, marjoram, and artificial honey on E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme (at a final concentration of 1% w/v), and the values were expressed as % (w/v) of undiluted honey.
Fig 1.
Representative TEM micrographs showing the structural changes in E. coli ATCC 8739 induced by honey treatment.
Log-phase E. coli cells were treated with 1× MIC concentrations of honey for 3 h at 37°C. Cells were harvested, glutaraldehyde-fixed, embedded in Epon 812 resin, examined using 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).
Fig 2.
Alterations in gene expression profiles associated with exposure of E. coli ATCC 8739 to the tested honeys as determined by qPCR.
Log-phase 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).
Fig 3.
Schematic diagram summarizing the effects of the tested honeys on E. coli ATCC 8739 at both structural and molecular levels.
In this figure, thin lines with pointed arrows indicate induction of gene expression or stimulation of a phenotype, while bar-headed lines indicate repression of gene expression or repression of a phenotype.