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. 2016 Mar 8;11(3):e0150984.
doi: 10.1371/journal.pone.0150984. eCollection 2016.

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli

Reham Wasfi  1 , Walid F Elkhatib  2 , Ahmed S Khairalla  3
Affiliations

Effects of Selected Egyptian Honeys on the Cellular Ultrastructure and the Gene Expression Profile of Escherichia coli

Reham Wasfi et al. PLoS One. .

Abstract

The purpose of this study was to: (i) evaluate the antibacterial activities of three Egyptian honeys collected from different floral sources (namely, citrus, clover, and marjoram) against Escherichia coli; (ii) investigate the effects of these honeys on bacterial ultrastructure; and (iii) assess the anti-virulence potential of these honeys, by examining their impacts on the expression of eight selected genes (involved in biofilm formation, quorum sensing, and stress survival) in the test organism. The minimum inhibitory concentration (MIC) of the honey samples against E. coli ATCC 8739 were assessed by the broth microdilution assay in the presence and absence of catalase enzyme. Impacts of the honeys on the cellular ultrastructure and the expression profiles of the selected genes of E. coli were examined using transmission electron microscopy (TEM) and quantitative real-time polymerase chain reaction (qPCR) analysis, respectively. The susceptibility tests showed promising antibacterial activities of all the tested honeys against E. coli. This was supported by the TEM observations, which revealed "ghost" cells lacking DNA, in addition to cells with increased vacuoles, and/or with irregular shrunken cytoplasm. Among the tested honeys, marjoram exhibited the highest total antibacterial activity and the highest levels of peroxide-dependent activity. The qPCR analysis showed that all honey-treated cells share a similar overall pattern of gene expression, with a trend toward reduced expression of the virulence genes of interest. Our results indicate that some varieties of the Egyptian honey have the potential to be effective inhibitor and virulence modulator of E. coli via multiple molecular targets.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
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
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
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.
See this image and copyright information in PMC

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