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Fructosazine: a Polyhydroxyalkylpyrazine with Antimicrobial Activity Mechanism of Inhibition against Extremely Heat-Resistant Escherichia coli Abhishek Bhattacherjee, Yuliya Hrynets, and Mirko Betti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03755 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 29, 2016

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Journal of Agricultural and Food Chemistry

Fructosazine: a Polyhydroxyalkylpyrazine with Antimicrobial Activity - Mechanism of Inhibition against Extremely Heat-Resistant Escherichia coli Abhishek Bhattacherjeea, Yuliya Hrynetsa and Mirko Bettia*

Affiliations a

Department of Agricultural, Food and Nutritional Science, University of Alberta

410 Agriculture/Forestry Centre Edmonton, AB T6G 2P5 Canada

*Corresponding Author Dr. M. Betti E-mail: [email protected] Tel: (780) 248-1598

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Abstract

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Fructosazine is a polyhydroxyalkylpyrazine recently reported to have antimicrobial activity

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against heat-resistant E. coli AW 1.7. This study investigated fructosazine’s antimicrobial

4

mechanism of action and compared it to that of riboflavin. Fructosazine–acetic acid was effective

5

in permeabilizing the outer membrane based on an evaluation of bacterial membrane integrity

6

using 1-N-phenyl-1-naphthylamine and propidium iodide. The uptake of fructosazine by E. coli

7

was pH-dependent with a greater uptake at pH 5 compared to pH 7 for all times throughout 16 h,

8

except 2, 3 and 10 h. Fructosazine generates 1O2, which is partially why it damages E. coli. DNA

9

fragmentation was confirmed by fluorescence microscopy and the fructosazine–acetic was the

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second most intense treatment after riboflavin-acetic acid. Electron microscopy revealed

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membrane structural damage by fructosazine at pH 5 and 7. This study provides evidences that

12

fructosazine exerts antimicrobial action by permeabilizing the cell membrane, damaging

13

membrane integrity, and fragmenting DNA.

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Key words: Fructosazine, Riboflavin, Heat-resistant E. coli, Singlet oxygen, Outer membrane

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permeabilization, DNA fragmentation

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Introduction

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Escherichia coli (E. coli) is a Gram-negative commensal bacterium which finds its optimal niche

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in the mucus layer of the mammalian colon. However, several E. coli clones have acquired

27

specific virulence attributes conferring an increased ability to adapt to new niches to cause a

28

broad spectrum of diseases. As a consequence, several “pathotypes” have emerged. For instance,

29

a subset of shiga toxin producing (STEC) pathotypes has been associated with hemorrhagic

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colitis and uremic syndrome.1, 2 In particular, O157:H7 is the most frequently isolated serotype

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of EHEC from ill people in the United States, Japan and the United Kingdom.3 The intestine of

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cattle is a major reservoir of STEC, and consumption of insufficiently cooked ground beef is

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associated with O157:H7 outbreaks worldwide. Specific virulent factors (i.e. the eae gene on the

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locus of enterocyte effacement) are responsible for colonization and adaptation of STEC in

35

intestines of cattle.4

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During slaughter, beef carcasses can be contaminated by STEC. Common post-mortem

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intervention strategies to control E. coli include thermal treatments, such as carcass steam

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pasteurization or hot water washes, as well as acid washes with 2-4% of lactic acid. Dlusskaya et

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al.5 isolated an extremely heat-resistant (but non-pathogenic) E. coli from a beef processing

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facility: the E. coli AW 1.7. This E. coli exhibits a D60 value for more than 60 min where most of

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the E. coli have D60 values below 1 minute. It can also survive in beef patties treated to a core

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temperature of 71°C. A specific 14 kb genomic island, the so-called locus of heat resistance, is

43

responsible for encoding proteins with putative function in cell envelope maintenance, turnover

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of misfolded proteins, and heat shock involved in the heat resistance of E. coli AW 1.7. This

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specific island was acquired through horizontal gene transfer.6 The ability of E. coli to evolve

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through horizontal gene acquisition of a specific genomic island makes the emergence of the

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pathogenic heat-resistant E. coli a possible risk; in other words, a “quantum leap” in E. coli 3 ACS Paragon Plus Environment


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evolution would be possible resulting in a bacteria strain that possesses both virulence and heat

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resistance.7,

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solutions to control and reduce harmful bacteria, including E. coli in meat products. The use of

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novel and alternative antimicrobial compounds is among the strategies available.9 These

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compounds include plant-derived essential oils, enzymes obtained from animal sources,

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bacteriocins from microbial sources, organic acids, and natural polymers like chitosan. In

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general, the modes of action of nearly all antimicrobials can be classified into one or more of the

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following groups: (i) reaction with the cell membrane, (ii) inactivation of essential enzymes, or

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(iii) destruction or inactivation of genetic material.10 For instance, plant essential oils (oregano,

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thyme and tea tree oils) or some of their phenolic constituents (carvacrol, eugenol and thymol)

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perturb the bacterial cell membrane,11, 12 whereas bacteriocins induce membrane pore formation

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and subsequent permeation of the cytoplasmatic membrane or inhibition of cell wall synthesis.13

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The use of weak organic acids (i.e., acetic acid) as a preservative has been used for thousands of

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years; here the undissociated forms of the acids pass through the plasma membrane by diffusion,

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liberate protons in the cytoplasm and lower the internal pH.14, 15, 16 Consequently, the substantial

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accumulation of the weak acid anions in the cytoplasm inhibits cell growth.17 These weak

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organic acids are also capable of affecting the integrity of the outer membrane (OM).17,

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Strategically designed combinations of these compounds - so called the “hurdle” technology -

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seem to be the key to successfully control pathogenic bacteria without relying on the traditional

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use of antibiotics, since they can lead to the development of bacterial resistance. Hrynets et al.19

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reported that glucosamine is an unstable molecule which degrades rapidly to form

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condensation products such as non-volatile polyhydroxyalkylpyrazines, the 2,5-bis(D-arabino-

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tetrahydroxybutyl)pyrazine (fructosazine, 1) (Figure 1) and 2-(D-arabino-tetrahydroxybutyl)-5-

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Both the food industry and scientific community are constantly looking for

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self-

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(D-erythro-2,3,4-trihydroxybutyl)pyrazine] (2,5-deoxyfructosazine), along with α-dicarbonyl

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compounds like 3-deoxyglucosone. Recently,20 it has been demonstrated that glucosamine

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caramel solution produced at 50°C also possesses antimicrobial activity and in addition to α-

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dicarbonyls can also generate acetic acid. The minimum inhibitory concentration (MIC)50 of 1

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against E. coli was 3.6 g/L at pH 5, while at pH 7 only 40% of the bacteria were inhibited at a

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plateau concentration of 6.6 g/L. Hence, a synergistic effect 1-organic acid was postulated to be

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important in generating a caramel solution with antimicrobial activity. The sublethal injury

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caused by the acetic acid to the OM of E. coli AW 1.7 may facilitate the permeation of 1 in the

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cytoplasm and induce damage to the bacterial DNA through the production of reactive oxygen

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species (ROS) (i.e., singlet oxygen). Indeed, singlet oxygen is generated during the glucosamine

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nonenzymatic browning.20 Compound 1 then might act similarly to the photosensitizer

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compound 2, riboflavin (Figure 1), a vitamin that can generate singlet oxygen and other radicals

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through a photoreactive process.21 Compound 2 alone or photodynamic processes involving the

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combined effect of 2 and UV light have been proposed to inactivate both Gram (-) and Gram (+)

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bacteria.22, 23

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Compound 1 is a flavor compound found in roasted food,24 while acetic acid is a taste

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contributor as well. Therefore, since glucosamine caramel is rich in these two chemical

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compounds,19, 20 its application might have the potential to increase the savouriness while at the

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same time inhibiting the growth of E. coli. The objective of this study was to determine the mode

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of action of 1 and a 1-acetic acid mixture on the inhibition of heat-resistant E. coli AW 1.7. The

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mechanism was evaluated by studying the integrity of the E. coli AW 1.7 membranes, and the

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uptake of 1 and bacteria DNA fragmentation. Microscopic examination was used to evaluate the



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effect of treatments on cell morphology. Compound 2, a known photosensitizer with

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antimicrobial activity, was used as a positive control.

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Materials and Methods

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Reagents and Chemicals

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Fructosazine, compound 1 (purity > 98%) was from Santa Cruz Biotechnology (Santa Cruz, CA,

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USA). The UHPLC profile and MS spectra of standard of 1 are given in Figure S1.

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Glutaraldehyde,

paraformaldehyde,

Polymyxin

B,

4-(2-hydroxyethyl)-1-

100

piperazineethanesulfonic acid (HEPES), 1-N-phenyl-1-naphthylamine (NPN) were from Sigma-

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Aldrich (St. Louis, MO, USA). NPN stock solution of 0.5 M was prepared in acetone and diluted

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to 40 µM in 5 mM HEPES buffer (pH 7.2). Propidium iodide (PI) was from BD Biosciences

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(San Jose, CA, USA). Singlet Oxygen Sensor Green (SOSG) reagent was from Thermo Fisher

104

(Waltham, MA, USA). HiPPR Detergent Removal Spin Column Kit was from Pierce (Thermo

105

Scientific, Canada). Micro-Halomax kit was obtained from Halotech DNA SL (Madrid, Spain).

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All chemicals were of analytical grade, and buffers were prepared with Milli-Q purified water

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(17 MΩ·cm, Millipore, Bedford, MA, USA).

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Bacterial Strain and Culturing Conditions

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Heat-resistant E. coli AW 1.7 was isolated from beef carcass and was grown on Difco Luria-

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Bertani (LB) medium supplemented with 15 g/L of agar.5 Final pH was adjusted to 7.0 ± 0.2 for

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both broth and agar media before autoclaving at 121°C for 20 min. Bacterial cells were incubated

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at 37°C under aerobic condition for 24 h and further used for microbial growth curve preparation

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and other microbial assays. The cells were cultivated in different experimental conditions and

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were abbreviated as follows: control (pH 7) - cells grown in LB medium at pH 7; acetic acid (pH

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5) - cells grown in LB medium at pH 5; 1 (pH 7) - cells grown in LB medium at pH 7 and


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supplemented with 3.6 g/L of fructosazine, 1-acetic acid (pH 5) - cells grown in LB medium at

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pH 5 and supplemented with 3.6 g/L of fructosazine, 2 (pH 7) - cells grown in LB medium at pH

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7 and supplemented with 3.6 g/L of riboflavin and 2-acetic acid (pH 5) - cells grown in LB

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medium at pH 5 and supplemented with 3.6 g/L of riboflavin. The LB medium was adjusted to

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pH 5 ± 0.2 using acetic acid.

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E. coli Growth Test

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Bacterial growth curves were obtained by a spectrophotometric method. Prior to microbial assay

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E. coli was aerobically subcultured twice in LB liquid medium at 37˚C for 24 h. Fresh bacterial

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culture (10 µL; 1 × 106 CFUs/mL, 1% inoculum) was mixed with 990 µL of LB medium (pH 5

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and 7 separately) to make a final volume of 1 mL. The initial optical densities at 630 nm (OD630)

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for all samples were standardized to 0.10 ± 0.04. Prior to microbial assay, compound 1 solution

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was sterile filtered (Millipore sterile 0.22 µM filter unit) and all the experiments were performed

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under aseptic conditions. The experimental samples were incubated at 37˚C up to 16 h (1 h

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interval from 0 to 10 h, 2 h interval from 10 to 16 h) and their OD630 were monitored over time

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using a plate reader (SpectraMax M3, Molecular Devices, Sunnyvale, CA, USA).

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Monitoring Single Oxygen by SOSG

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In vitro 1O2 production was monitored by SOSG reagent.25 The fluorescence of SOSG is

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quenched in its intact form and produces strong fluorescence after 1O2 oxidation.26 1 was

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incubated at 37˚C at a concentration of 3.6 mg/mL and collected at 0, 4, 8, 12 and 16 h. Fresh

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SOSG stock (5 mM) was prepared by adding 33 µL methanol to a 100 µg vial. Because SOSG is

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sensitive to light, the analyses were carried out in the dark. SOSG at 10 µM final concentration

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was added to collected samples and mixtures were incubated for 15 min. Fluorescence was

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detected at 525 nm (λexc = 504 nm). An SOSG blank was produced and tested under identical

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conditions. Its emission intensity was low, showing that the emission of SOSG fluorescence was 7 ACS Paragon Plus Environment


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not due the light-induced fluorescence of SOSG. Followed subtraction of the emission intensity

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of the control sample from the emission intensities of the sample values, the data were fitted with

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a nonlinear regression using GraphPad Prism software. Compound 2 was used as a positive

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control and tested under the same conditions as 1.

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Determination of the Intracellular Singlet Oxygen

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Bacterial cultures were maintained according to the experimental conditions as mentioned in the

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earlier bacterial growth section. Bacterial cell suspensions were collected from all treatments

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over time and were pelleted by centrifugation at 1100 rpm for 10 min. The recovered cell pellet

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was collected and washed three times with PBS and re-suspended in PBS (pH 7.4). The bacterial

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solution was treated with ice-cold RIPA buffer and agitated for 20 min at 4°C. The suspension

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was further sonicated and centrifuged at 12000 rpm at 4°C for 20 min. The supernatant

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containing the bacterial intracellular content was collected and presence of 1O2 was monitored by

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SOSG probe as mentioned in the previous section.

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Membrane Permeabilizaion Assays

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Lipopolysaccharide (LPS), or endotoxin, of OM in Gram (-) bacteria acts as a protective

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permeability barrier. LPS is cross-bridged to the peptidoglycan of the cell wall27 and the damage

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to the cell wall integrity consequently affects also LPS integrity.28 To evaluate the permeability

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of E. coli’s AW 1.7 membrane due to exposure to 1 at pHs 5 and 7 two assays were used: i) the

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OM permeability by using NPN uptake assay and ii) uptake of extracellular PI by permeabilized

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cells.

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Outer Membrane (OM) Permeability Test

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Bacterial cells were collected at different time points and centrifuged at 2000 rpm for 5 min. Cell

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pellet was collected and washed twice with PBS buffer. After washing, cell pellet was suspended


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in 5 mM HEPES buffer (pH 7.4) supplemented with 5 mM glucose. Aliquots of this cell

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suspension (200 µL) were pipetted into black fluoroplate wells, which contained 10 µM NPN.

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An increase in fluorescence emission due to partitioning of NPN into OM was monitored at 420

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nm (λexc = 350 nm) using a SpectraMax M3 plate reader. Each sample was monitored within 2

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min and fluorescence readings were taken after the values stabilized. Polymyxin B was used as a

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positive control due to its strong OM permeabilizing properties. Briefly, bacterial cells were

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treated with 10 µg/mL Polymyxin B and incubated for 1 h at 37°C. After incubation period NPN

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was added and fluorescence was measured as mentioned above. Fluorescence of Polymyxin B

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treated sample was designated as F100 or positive control. Experiment control was tested at 0 time

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point with E. coli (cultured at pH 7) and NPN together to monitor background interference and

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this value was designated as F0 or initial fluorescence. NPN fluorescence in all other

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experimental treatments was marked as Fobs or observed fluorescence. Percentage of NPN uptake

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was calculated by the following formula: % NPN uptake = (Fobs – F0)/(F100-F0) × 100.

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Propidium Iodide (PI) Uptake Assay

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The experiments were performed using the non-pathogenic E. coli strain cultured as described in

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the previous section. Flow cytometric analyses were performed according to Park et al.29

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Bacterial cells were collected at 0, 4, 8, 12 and 16 h. To minimize clumping and stabilize cell

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structures, collected cells were fixed with a solution of 2.5% glutaraldehyde and 2%

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paraformaldehyde in 0.1 M phosphate buffer for 30 min at 4°C. Cells were then pelleted by

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centrifugation at 2000 rpm for 5 min, washed and resuspended in phosphate buffer saline (PBS).

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The cells were stained with a membrane-impermeable red-fluorescent nucleic acid stain, PI, to

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detect nonviable cells. The experimental procedure for staining involved adding PI to the

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bacterial samples to a final concentration of 10 µg/mL. The sample was then vortexed before



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incubating in the dark for 30 min at 4˚C. Fluorescence of PI was achieved via excitation at 488

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nm with emission at 610/20 nm band pass filter. Cells sensitive to PI were detected using a BD

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LSR Fortessa X-20 flow cytometer (BD Biosciences, San Jose, CA, USA) and

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histograms/scattered plots were generated by BD FACSDiva 8.0.1 software.

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Fructosazine Uptake by E. coli AW 1.7

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Bacterial cell suspensions (500 µL containing 1 × 106 CFUs) were collected from all treatments

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over time. The cultures were then pelleted by centrifugation at 1100 rpm for 10 min. The

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recovered cell pellet was collected, whereas the supernatant was discarded. Bacterial cells were

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further washed three times with PBS and re-suspended in PBS (pH 7.4). Bacterial solution was

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treated with ice-cold RIPA buffer (0.15 mM NaCl/0.05 mM Tris·HCl, pH 7.2/1% Triton X-

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100/1% sodium deoxycholate/0.1% SDS) and agitated for 20 min at 4°C. The suspension was

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further sonicated for 30 min and centrifuged at 12,000 rpm at 4°C for 20 min. Supernatant

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containing the bacterial intracellular content was collected and residual detergents were removed

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by HiPPR detergent removal spin columns. Samples were subjected to analyses using an

200

UHPLC system (Shimadzu, Columbia, MD, USA). Ten microliters were injected and separated

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on an Ascentis Express ES-C18 column (Sigma-Aldrich) using conditions described before.20

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Quantitation was performed by using an external calibration curve built with a set of seven

203

standard dilutions of 1 standard. The average limits of detection (LODs) and limits of

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quantitation (LOQs) were 1.42 ± 0.02 and 4.69 ± 0.07 µg/mL, respectively. The calibration curve

205

was linear with a correlation coefficient of 0.999. Each data point was analyzed in triplicate. To

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ensure correct peak identification, in addition to the same retention time as standard 1, peaks

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were collected and subjected to mass spectrometry analyses using a 1200 series HPLC unit

208

(Agilent, Palo Alto, CA, USA) connected to a 4000 Q TRAP LC-MS/MS System (Applied


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Biosystems, Concord, ON, Canada). The HPLC separation was performed under exactly the

210

same as described above for UHPLC. The conditions of analyses using electrospray ionization in

211

a positive mode the same as described in Hrynets et al.20

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Determination of DNA Fragmentation

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The commercial Micro-Halomax kit was used for bacterial DNA fragmentation analysis.

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Experimental samples (45 µL) were collected at 16 h from each treatment and mixed gently

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using a pipette with 100 µL of melted agarose. Cell suspensions (5 µL) – agarose mixture were

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placed onto the pre-coated agarose slides provided by the manufacturer and covered by 18×18

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mm cover slip. Slides were placed horizontally on a pre-cooled (at 4˚C) glass plate and kept at

218

4˚C for 5 min. After the incubation time slides were removed from the fridge and cover slip was

219

removed carefully. The slides were immersed in 10 mL of lysis solution (pre-heated at 37˚C) for

220

5 min. Followed incubation slides were washed in distilled water, dehydrated in sequential 70,

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90 and 100% ethanol baths for 3 min in each gradation. Slides were then dried at a room

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temperature and incubated in an oven at 80˚C for 1 h. Slides were stained with 100 µL of SYBR

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Gold (diluted 1:200 in TBE buffer, Invitrogen). Fluorescence microscopy images were taken

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using a Leica DMX RA microscope immediately after staining to avoid drying. Small nucleoids

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with fragmented DNA were assed under 100X objective lense and Q Capture Pro7 software was

226

used to analyze the images. Digital image analyses were performed according to Fernández et

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al.30 using Visilog 7.3 software (Noesis, Gif sur Yvette, France).

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Scanning Electron Microscopy (SEM)

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The morphological changes of bacterial cells treated with or without 1 at pHs 5 or 7 were

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investigated by SEM. Bacterial samples at 16 h were fixed with 2.5% glutaraldehyde, 2%

231

paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 4 h at a room temperature. After the



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fixation period, fixative was drained off and cells were gently washed with 0.1 M phosphate

233

buffer (pH 7.0) for 3 times (15 min each). Fixed specimens were dehydrated through a series of

234

ethanol solutions of increasing concentration (50, 70, 90, and 100%, v/v) for 15 min each. After

235

dehydration, each specimen was dried with hexamethyldisilazane (HMDS).31 One hundred

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percent ethanol samples were transferred to ethanol:HMDS (75:25), ethanol:HMDS (50:50),

237

ethanol:HMDS (25:75) and HMDS (100%) gradually for 20-30 min each. After each step

238

samples were centrifuged (2500 rpm, 5 min) and pellet was collected. Finally HMDS (100%)

239

was poured off and all the samples were left to evaporate overnight. Specimen were mounted on

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SEM stubs, sputter-coated with Au/Pd using a Hummer sputtering system (Anatech Ltd., Battle

241

Creek, MI, USA) and imaged using a Philips/FEI (XL 30) scanning electron microscope

242

(Philips/FEI, Hillsboro, OR, USA) with an electron bean energy of 20 kV. The images were

243

processed by Scandium 5.0 software.

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Transmission Electron Microscopy (TEM)

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Bacterial samples were collected from all treatments at 16 h. The samples were fixed with 2.5%

246

glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 4 h at room

247

temperature. After fixation period, fixative was drained off and cells were washed (3 times) with

248

0.1 M phosphate buffer. The buffer was drained off and samples were post fixed with 1%

249

osmium tetrachloride (OsCl4 in 0.1 M buffer, pH 7.2) for 1.5 h. After post fixation, samples were

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washed with 0.1 M phosphate buffer 3 × 15 min each followed by dehydration in graded ethanol

251

series (50, 70, 90 and 100%) for 15 min each. Samples were mixed with at a 1:1 ratio with

252

ethanol and Spurr’s resin and kept for 3 h. After incubation, ethanol-Spurr’s resin solution was

253

removed and replaced by a pure Spurr’s resin solution. Experimental tubes were kept in an oven

254

at 70-80˚C overnight (16-18 h). Solidified samples were removed from the oven and cooled to


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room temperature. Ultramicrotome (Ultracut E model, Reichert-Jung, Austria) equipped with a

256

diamond blade was used for ultrathin thickness (80-100 nm) sectioning. Sections obtained from

257

microtome were stained with 2% uranyl acetate and 2% lead citrate. The specimens were

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mounted on Formvar and carbon-coated 300 mesh copper grids (Ted Pella Inc., Redding, CA,

259

USA) and examined with a transmission electron microscope (TEM) (Fei-Philips Morgagni 268,

260

Fei Company, Hillsboro, OR, USA) operating at 80 kV. High resolution (8.9 and 56 kV)

261

micrographs were taken using a charge-coupled device camera and controller (Gatan Orius,

262

Pleasanton, CA, USA) and processed using DigitalMicrograph software.

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Statistical Analysis

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Each treatment was performed in triplicate (n = 3). Analyses were performed using SAS

265

software (v. 9.3, SAS Institute, Cary, NC, USA). Comparisons of intracellular content of 1O2, PI-

266

stained cells, NPN uptake among treatments within the same time frame were performed using

267

analysis of variance (ANOVA). Significant differences were identified by the post hoc Tukey’s

268

test at a p value of less than 0.05. Comparisons between pH-dependent 1 uptake at specific time

269

points were performed using a t test. Results of E. coli growth and 1O2 generation were fitted in

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Graph Pad Prism software (v. 7, San Diego, CA, USA) using a nonlinear curve-fitting model

271

with the following equation: Y = Y0 + (Ymax − Y0) × (1 − exp[−Kx]). The quantitative data are

272

presented as the mean ± standard deviation of the mean.

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Results and Discussion

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Effect of the Treatments on the Growth of E. coli AW 1.7

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As highlighted in the introduction, it is hypothesized that 1 exerts its antimicrobial activity with a

276

mechanism similar to the photosensitizer 2. Hence, in addition to 1 (pH 7), acetic acid (pH 5) and

277

1-acetic acid (pH 5) treatments, compound 2 positive controls with or without acetic acid (pH 5)



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were also necessary in this study. As shown in Figure 2A, a difference among treatments started

279

to be noticeable at 6 h of growth and this became clearer by the end of the growth at 16 h. OD

280

values of 1.0, 0.56, 0.64, 0.48, 0.57 and 0.40 for the negative control (pH 7), acetic acid (pH 5), 1

281

(pH 7), 1-acetic acid (pH 5), 2 (pH 7) and 2-acetic acid (pH 5) were found at 16 h which

282

corresponded to a 46, 39, 54, 47 and 64% of growth inhibition as compared to the control,

283

respectively (Figure S2). In the previous study20 1 reached to MIC50 at pH 5 at 3.6 g/L.

284

Accordingly, 1-acetic acid treatment caused a 52% inhibition at the same concentration. Acetic

285

acid and 1 treatments alone also showed effect on E. coli growth inhibiting 46 and 39%,

286

respectively. When the combination of 1 and acetic acid was used an additional 15% inhibition

287

against E. coli was gained as compared to the treatment containing 1 alone, however the

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mechanism of action of this combination is not likely synergistic as it was originally

289

hypothesized.20 The positive control 2-acetic (pH 5) was able to inhibit 64% of E. coli growth,

290

which was 10% higher than the 1-acetic acid treatment.

291

In general, appreciable inhibitory effects were observed from both acetic acid and 1 when used

292

alone against E. coli. The mode of inhibition of acetic acid on E. coli has been attributed to: i)

293

changes in cytoplasmatic pH and energy status of the cell; ii) perturbation of the membrane

294

function; iii) accumulation of acetate anions within the cells. In the latter case, Roe et al.18

295

indicated that the ability of acetate to inhibit E. coli growth arises from the depletion of the

296

intracellular methionine pool with the concomitant accumulation of the toxic intermediate

297

homocysteine; this would further augment the effect of lowering cytoplasmic pH. Dlusskaya et

298

al.5 reported that the acquisition of the heat resistance by E. coli AW 1.7 makes this strain more

299

susceptible to an acidic environment compared to the non-resistant E. coli strains. The heat

300

resistant property of E. coli AW 1.7 is linked to the overexpression of the porin NmpC at 37°C,


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which is not expressed in the majority of E. coli strains.32 In general, E. coli’s trimeric porins

302

(OmpC, OmpF and PhoE) are responsible for the passage of small hydrophilic molecules

303

through the OM.33 Therefore it would be of interest to study if the overexpression of certain

304

porins (i.e. NmpC) is related to this increased susceptibility towards organic acids.34 Porins may

305

also be involved in the uptake of 1. Compound 1 molecules are relatively small and amphiphilic

306

with a MW of 320 Da. Hence they are also candidate solutes to be moved along the porins in

307

Gram (-) bacteria. Interestingly, when β-carotene, a quencher of 1O2 and other ROS, was added

308

to the treatments, the inhibitory capacities of both 1 and 2 were greatly reduced (Figure S3A, B).

309

This implies that 1O2 or other ROS may be responsible for the antimicrobial property of 1. These

310

results prove that 1 alone, a product of glucosamine’s non-enzymatic browning, possesses some

311

antimicrobial activity. To date, the only Maillard reaction products with proven antimicrobial

312

activities are high MW (> 10 kDa) melanoidins isolated from coffee and biscuits35 and

313

aminoreductones36,

314

mechanism of melanoidins have been linked to their capacity to destabilize the OM through the

315

chelation of important divalent cations like Mg++,36,37 the mechanism of action of the

316

aminoreductones has not yet been fully elucidated. Since the generation of ROS seems to be the

317

key for 1 antimicrobial activity, the capacity of different treatments to produce 1O2 will be

318

discussed in the next section.

319

Singlet Oxygen Production from Fructosazine in vitro and within E. coli AW 1.7

320

The production of 1O2 during the Maillard reaction has been documented.38 Melanoidins, for

321

instance, are one of the important compounds of the Maillard reaction products that can generate

322

1

323

browning conducted at 50°C was also shown to generate 1O2.20 Hence, 1 was hypothesized to

37

produced during the early stage of the Maillard reaction. Although the

O2 from triplet ground oxygen, thus acting as a photosensitizer.39 Glucosamine nonenzymatic



Page 16 of 42

324

promote 1O2 production. Supporting this hypothesis is the work of Markham and Sammes.40

325

These authors discovered that pyrazines can form endo-peroxide compounds through the 1,4-

326

addition of 1O2 across two double bonds in the pyrazine molecule even without the use of the

327

photosensitizer Methylene Blue, suggesting that the pyrazines can act as their own

328

photosensitizer. Singlet oxygen is a strong oxidizing reagent which can impair bacterial

329

metabolism and DNA. To determine whether 1 have the potential to generate 1O2 and thus alter

330

E. coli metabolism, an in vitro experiment was conducted using the fluorescent probe SOSG to

331

trap 1O2 (Figure 2B). In this study, 2 (vitamin B2) was used as a control as it is an efficient

332

photosensitizer capable of inducing oxidative damage to light-exposed tissue and food.

333

Compound 2 may transfer the excitation energy to ground-state triplet oxygen in a spin-allowed

334

process to yield 1O2.41 Figure 2B shows that incubation time had a significant effect on the

335

progress of 1O2 generation from 0 to 12 h, with no difference from 12 to 16 h. No major effect

336

between pHs 5 and 7 was also found for 2. However, compound 2 fluorescence from 4-16 h was

337

on average a 3.7-fold greater compared to 1. Hence, 1 seems to play a significant role in the

338

production of 1O2 in glucosamine nonenzymatic browning and it is plausible to assume that the

339

amount of ROS produced play a role in inhibiting E. coli growth. To best of our knowledge, this

340

is a first time that direct evidence of 1O2 generation from a hydroxyalkylpyrazine has been

341

provided.

342

To confirm whether upon intracellular uptake 1 generates 1O2, and thus causing damage from

343

within the cell, the intracellular 1O2 was determined and the results are shown in Figure 2C.

344

Significant differences among treatments were observed at each time point, except 0, with the

345

highest 1O2 generation in the 2 at longer times, followed by 1-acetic acid (pH 5) and 1 (pH 7).

346

Compound 1 at pH 7 produced a significant amount of 1O2 over time, and a relatively small


Page 17 of 42


347

(14.7% on average) additional production of 1O2 was obtained when the mixture 1-acetic acid

348

was used. Despite the major contribution of 1O2 from 1, acetic acid used alone was also shown to

349

generate this ROS, particularly at a longer incubation time (16 h), implying the link between acid

350

and oxidative stress.42 Singlet oxygen is a highly reactive form of molecular oxygen that initiates

351

oxidative reactions in the bacterial cell wall, lipid membranes, enzymes, or nucleic acids harming

352

living systems.43 Sies44 reported that 1O2 selectively reacts with the deoxyguanosine moiety in

353

DNA causing strand breaks in DNA, as studied in plasmids and bacteriophages. Singlet oxygen

354

is also capable to induce a permeabilization effect on lipid membranes,45 thus promoting uptake

355

of 1. However, when β-carotene was added to the 1 and 2, the production of 1O2 from E. coli was

356

strongly reduced (Figure S4A, B). This implies that the major source of 1O2 produced within E.

357

coli cells come from 1 or 2 molecules. However, the possibility cannot be excluded of a type I

358

photosensitization within which triplet 1 reacts directly with substrate molecules (i.e., DNA).

359

Effects of the Treatments on E. coli AW 1.7 Membranes Permeabilization

360

Outer Membrane Permeabilization Estimated by NPN Uptake

361

The first step of any antimicrobial compound to be effective against Gram (-) bacterium is to be

362

able to penetrate their negatively charged OM. This means that the antimicrobial agents have to

363

possess a certain affinity for the porins, or be able to induce pore formation or affect the integrity

364

of the OM by a direct disruptive action against the negatively charged LPS. NPN is a neutral

365

hydrophobic fluorescence probe which is generally excluded by bacterial OM. An increment in

366

NPN fluorescence suggests its penetration inside of the OM due to its damage.46 Therefore the

367

NPN uptake assay can be used as an indicator to measure OM permeability. The results of the

368

NPN uptake experiments show that after 16 h of incubation, the fluorescence emission of NPN

369

dye was 2948, 4443, 4218, 5000, 4772 and 6078 for control (pH 7), acetic acid (pH 5), 1 (pH 7),



Page 18 of 42

370

1-acetic acid (pH 5), 2 (pH 7) and 2-acetic acid (pH 5), respectively (Figure S5). Compound 1-

371

acetic acid treatment showed a greater NPN uptake of 49.4% compared to acetic acid and 1

372

alone, which were 36.2 and 30.9, respectively (Figure 3A). However, the 2 (positive controls)

373

were 74.6 and 43.5% for NPN uptake at pH 5 and 7, respectively. This indicates that a 1-acetic

374

acid combination has a damaging effect on the OM; however the 2-acetic acid treatment was

375

more effective. Compound 1 alone at pH 7 caused a 30.9% uptake of NPN, implying a

376

permeabilizing effect for 1 alone. This is the first time a publication has reported the damaging

377

effect of 1 on E. coli’s OM. While it is known how acetic acid damages the OM of E. coli,47 the

378

permeabilization effect of 1 on E. coli’s OM is a new discovery. Kotova et al.45 reported a

379

photodynamic mechanism in which 1O2 induces a permeabilization of lipid membranes via

380

oxidation of polyunsaturated fatty acids. The results obtained in this study indicate that with

381

greater amounts of 1O2 produced, there is more OM damage. Indeed, when β-carotene was added

382

to the 1 or 2 treatments, OM damage was significantly reduced, confirming the direct role of 1O2

383

or other ROS in the perturbation of E. coli’s OM (Figure S6A, B). Although the combination of

384

1-acetic acid or 2-acetic acid were the most effective in damaging E. coli’s OM, the mechanism

385

is likely not synergistic. It is possible that under acid stress E. coli expresses antioxidant enzymes

386

to counterattack the formation of ROS.42

387

Cellular Membrane Integrity by PI Staining

388

To further access the cell permeability, a deoxyribonucleic acid (DNA) intercalating fluorescent

389

dye, PI, was applied and visualized by flow cytometry. PI can enter the E. coli cell only when the

390

membrane is permeabilized and stoichiometrically binds to cell’s DNA.48 Based on NPN assay,

391

acetic acid and 1 treatments caused E. coli’s cell OM permeability, therefore it is likely that free

392

diffusion of small dyes such as PI into cytoplasm occurs. In this scenario, the increment in PI


Page 19 of 42


393

fluorescence emission is proportional to the amount of internalized PI and indicative of

394

compromised cell membrane permeability of disintegrating cells.26 Bacterial fluorescence

395

distribution histograms obtained for the PI uptake by E. coli AW 1.7 cells are presented in Figure

396

S7A, B, where the treated cells showed a shift of fluorescence peak towards right. The

397

quantitative evaluations of flow cytometry analyses (Figure 3B) showed an increase in PI-stained

398

cells over time with the most significant effect of 2-acetic acid (pH 5) treatment. At 16 h of

399

treatments 2.6, 44.4, 38.7, 51.7, 47.7 and 64.0% of the cells were PI-stained in control, acetic

400

acid, 1 (pH 7), 1-acetic acid (pH 5), 2 (pH 7) and 2-acetic acid (pH 5), respectively. In

401

accordance to the E. coli growth curves (Figure 2A), a greater percent of the PI uptake (51.7%)

402

was found in the 1-acetic acid treatment compared to 1 (pH 7) or acetic acid used alone. Once

403

again, this confirms that growth of bacterial cultures at pH 5 in the presence of 1 results in a

404

greater level of damage of E. coli AW 1.7. Remarkably, at 8 and 12 h no statistical difference

405

was found between the acetic acid (pH 5) and 1 treatments, suggesting that the effect of 1 on the

406

cell permeability is comparable to that of acetic acid at pH 5. Hence, compound 1 not only has

407

the capacity of damaging the OM, but also affects the inner membrane and thus penetrates into

408

the cytoplasm. When β-carotene was added to the treatments, a dramatic reduction of the PI into

409

cytoplasm was observed (Figure S7B, S8).

410

Fructosazine Uptake by E. coli

411

In an earlier study20 the presence of 1 inside E. coli AW 1.7 strain was demonstrated by using

412

mass spectrometry. However, no quantitation was attempted at that time. To test the extent of 1

413

uptake by E. coli in relation to the treatments, a cell-free intercellular extract “soup” was

414

collected at different time points and injected into UHPLC. The retention time of the major peak

415

in bacterial samples corresponded to that of an authentic standard of 1 and eluted at 3.5 min



Page 20 of 42

416

(Figure 4A). Collision-induced fragment of potassium-cationized 1 was found at m/z 359.4

417

(Figure 4B). The presence of potassium ions is likely from the sample itself, since K+ is the

418

major monovalent intracellular cation of E. coli.49 When a precursor ion at m/z 321.7 was

419

fragmented, the fragmentation pattern was the same as reported for 1 previously20 and

420

characterized by the loss of one and two water molecules (Figure 4C). Confirmation of 1

421

presence in the collected bacterial samples allowed for quantitative estimation. As shown in

422

Figure 4D, the percent of 1 uptake was plotted as a function of incubation time at pHs 5 with

423

acetic acid or 7 (control). The effect of pH on 1 uptake was evident at all time points tested

424

except 2, 3 and 10 h of incubation. For the other times, the uptake of 1 was significantly greater

425

at pH 5. The greatest uptake of 1 at this pH was found at 5 h, followed by a decrease at 6 h. A

426

different uptake pattern over time was found at pH 7, where no difference in 1 concentration was

427

obtained from 5 to 14 h. In general, the 1 uptake was time-dependent with a decrease from

428

around 10 to 16 h. These results agree with the tests of membrane permeability (Figure 3A, B),

429

where more pronounced damaging effect was found at pH 5. However, appreciable 1 uptake was

430

also evident at pH 7, probably due to the ability of 1 to be moved along the porins and due to

431

their permeabilizing effect as previously reported. These results indicate the 1 was found within

432

E. coli AW 1.7 cytoplasm and thus it possible that 1 generates ROS that can impair the E. coli’s

433

membranes and induce DNA fragmentation.50

434

Fragmentation of E. coli’s AW 1.7 DNA in Response to the Treatments

435

The method reported by Santiso et al.51 is an effective way to evaluate DNA fragmentation in

436

bacteria. In this methodology bacteria are immersed in an inert microgel on a microscope slide

437

and incubated in a specific lysis solution that removes the cell wall, membranes and proteins

438

leaving only the bacteria DNA attached to the microgel. However, if the DNA is fragmented as a


Page 21 of 42


439

consequence of an antimicrobial treatment, haloes (nucleoid spread) of the peripheral diffusion

440

residual central core are visualized under fluorescence microscopy after staining with a sensitive

441

fluorochrome.51 In general, the greater the nucleoid spread implies the greater the DNA

442

fragmentation. The effect of 1 or 1-acetic acid mixture as well as 2 on DNA integrity is presented

443

in Figure 5. As described by Santiso et al.51 control cells (pH 7) do not spread their nucleoid. In

444

this study, a restricted spreading of nucleoid was observed in a control (pH 7) treatment (Figure

445

5A a), which can be due to the background effect of the lysis solution. Treatments with acetic

446

acid (pH 5) and 1 (pH 7) showed a greater nucleoid spread (Figure 5A b, c) compared to the

447

control. The combination of 1-acetic acid (pH 5) resulted in larger haloes compared to 1 and

448

acetic acid used alone (Figure 5A d). The strongest nucleoid spread was observed for treatments

449

2 (pH 7) and 2-acetic acid (pH 5) (Figure 5A e, f). Quantitative analysis of digital images (Figure

450

5B) revealed the greatest fragmentation in the 2-acetic acid (pH 5) treatment, followed by 1–

451

acetic acid (pH 5). In general, the 1–acetic acid (pH 5) treatment was comparable to 5 µg/mL

452

ciprofloxin, a fluoroquinone that induces DNA double-strand breaks.52 This means that all of the

453

treatments were able to induce DNA fragmentation, and that the combination of 1 and acetic acid

454

produced a significant DNA fragmentation comparable to the results obtained in the previous

455

section regarding 1O2. The addition of β-carotene to 1 (pH 7) and 1-acetic acid (pH 5) reduced

456

the mean surface area of nucleoids by 5.7 and 1.4 times, respectively (Figure S9A, B). However,

457

as mentioned in previous section, a type I photosensitization where triplet 1 reacts directly with

458

DNA can also be possible.

459

Bacterial Morphology Evaluated by Electron Microscopy

460

To have more insights about the mode of antimicrobial action of 1, acetic acid and 1-acetic acid

461

treatments the morphology of E. coli Aw 1.7 was studied by SEM. The morphology of the



Page 22 of 42

462

bacteria is shown in Figure 6A and cells from stationary phase (16 h) were evaluated. In general,

463

all the treatments dramatically altered the morphology of E. coli cells in comparison to the

464

control treatment grown at pH 7 without any antimicrobials, which was represented by bacteria

465

of a regular surface morphology typical for E. coli with uniform appearance. Severe alterations

466

to the bacterial morphology, consistent with a wrinkled surface and slight blebbing, were evident

467

in acetic acid, 1 and 1-acetic acid treatments.

468

TEM was employed to evaluate the integrity of the bacterial cell membrane and intercellular

469

alterations of E. coli Aw 1.7 with results shown in Figure 6B. The control treatment represents

470

TEM images of a defined cell wall with a dense protoplasm and well-organized smooth

471

membrane and clearly defined thin periplasmic space. An evenly stained cell interior also

472

corresponds to the presence of DNA and proteins. Two major significant changes can be

473

observed in 1, acetic acid and 1-acetic treatments described by i) sunken protoplasm near the

474

edges of bacterial cell wall evident by low density regions near the membrane and ii) irregular-

475

rough outer membranes with dislocated inter cellular content. The most disrupted E. coli

476

membrane seems was observed in the 1-acetic acid treatment in accordance to the greatest

477

percentage of bacterial cell permeability (Figures 3A, B) and DNA fragmentation (Figure 5).

478

Compound 1 treatment at pH 7 shows relatively less damaged cells; however rough membrane

479

and dislocated cellular content were evident. This suggests that 1 alone can affect the

480

morphology of E. coli AW 1.7 and corroborates the results described in Figures 3A and B

481

reporting NPN and PI uptake, indicating increased membrane permeability. This is an important

482

finding since it likely indicates that 1 alone has a similar damaging effect on the E. coli’s

483

membranes as does acetic acid at pH 5. This latter opens the possibility of exploiting the the

484

permeabilization effect of 1 to promote a concerted inhibitory action with other antimicrobials


Page 23 of 42


485

(i.e. bacteriocine) against E. coli, and possibly other pathogenic Gram (-) bacteria, particularly in

486

those food products where the acidity is not favourable.

487

In conclusion, the findings reported in this study elucidate the mechanism of action of 1 and 1-

488

acetic acid mixture against E. coli AW 1.7. The basic mechanism of 1 is a photoreactive process

489

in which the photosynthetizer 1 produces 1O2 with subsequent negative effects on bacterial

490

membranes and DNA, similar to that of the 2. Despite not being synergistic, this effect is

491

amplified in acidic conditions at pH 5 due to the effect of acetic acid on E. coli’s OM

492

permeabilization. This research also confirms that nonenzymatic browning reactions can be a

493

source of valuable compounds possessing both flavor and antimicrobial activity. In order to find

494

an effective and synergistic inhibition mechanism, studies combining 1 and UV light as well 1

495

and other antimicrobial compounds are currently being evaluated.

496

Abbreviations Used

497

ANOVA, analysis of variance; DNA, deoxyribonucleic acid; E. coli, Escherichia coli; HMDS,

498

hexamethyldisilazane;

499

lipopolysaccharide;

500

naphthylamine; PI, propidium iodide; ROS, reactive oxygen species; SD, standard deviation;

501

SEM, scanning electron microscopy; SOSG, singlet oxygen sensor green; TEM, transmission

502

electron microscopy; UHPLC, ultrahigh performance liquid chromatography.

503

Acknowledgments

504

We thank Dr. Maurice Ndagijimana for helping with antimicrobial assays and Dr. Michael

505

Gänzle (Department of AFNS, University of Alberta) for providing E. coli Aw 1.7. We also

506

thank Dr. Aja Rieger from the Faculty of Medicine and Dentistry Flow Cytometry Facility

507

(University of Alberta) for the assistance with flow cytometry analyses and Arlene Oatway from

LOD, MIC,

limit

of detection;

minimum

inhibitory

LOQ,

limit

concentration;

of quantitation; NPN,

LPS,

1-N-phenyl-1-



Page 24 of 42

508

the Department of Biological Sciences, Advanced Microscopy Facility (University Of Alberta)

509

for the assistance with electron microscopy.

510

Funding Sources

511

This research was funded by grant from Alberta Livestock Meat Agency (ALMA), Alberta

512

Innovates – Bio Solutions (Al Bio), and Natural Sciences and Engineering Research Council of

513

Canada (NSERC).

514

Supporting Information

515

Figure S1. Characterization of fructosazine (compound 1) standard. Figures S2 and S3.

516

Percentages of inhibition of E. coli AW 1.7 exposed to different experimental treatments; Figure

517

S4. SOSG fluorescence as an indicator of 1O2 formation by 1 or 2 in the presence of β-carotene;

518

Figure S5. Fluorescence emission intensity of NPN by E. coli AW 1.7 exposed to different

519

experimental treatments; Figure S6. Fluorescence emission intensity of NPN and NPN uptake

520

(%) by E. coli AW 1.7 exposed to different experimental treatments; Figure S7. Histograms of E.

521

coli AW 1.7 exposed to different experimental treatments; Figure S8. Membrane

522

permeabilization of E. coli AW 1.7 measured by percentage of PI uptake as quantitated from

523

flow cytometry data. Figure S9. Fluorescence micrographs and digital image analyses

524

corresponding to the DNA fragmentation of E. coli AW 1.7 exposed to different experimental

525

treatments.

526

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527

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2008, 56, 2357−2362.

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[36] Trang, V. H.; Takeuchi, H.; Kudo, H.; Katsuno, S.; Shimamura, T.; Kashiwagi, T.; Hong

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pathogenic bacteria methicillin-resistant Staphylococcus aureus (MRSA). J. Agric. Food

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Chem. 2011, 59, 8953−8960.

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[37] Trang, V. H.; Thanh, L. X.; Sarter, S.; Shimamura, T.; Takeuchi, H. Study of the

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antimicrobial activity of aminoreductone against pathogenic bacteria in comparison with

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other antibiotics. Int. J. Biol. Biomol. Agric. Food Biotechnol. Eng. 2013, 7, 1131−1134.

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[38] Hiramoto, K.; Kato, T.; Kikugawa, K. Generation of DNA-breaking activity in the Maillard

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reaction of glucose-amino acid mixtures in a solid system. Mutat. Res. 1993, 285, 191-198. [39] Argirova, M. D. Photosensitizer activity of model melanoidins. J.Agric. Food Chem. 2005, 53, 1210−1214. [40] Markham, J. L.; Sammes, P. G. Oxygenation of pyrazines and pyrimidines. J. Chem. Soc., Chem. Commun. 1976, 417-418. [41] Cardoso, D. R.; Libardi, S. H.; Skibsted, L. H. Riboflavin as a photosensitizer. Effects on human health and food quality. Food Funct. 2012, 3, 487-502.

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[42] Bruno-Barcena, J. M.; Andrus, J. M.; Libby, S. L.; Klaenhammer T. R.; Hassan H. M. Role

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of antioxidant enzymes in bacterial resistance to organic acids. Appl. Environ. Microbiol.

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2010, 76, 2747-2753.

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[43] Maisch, T.; Baier, J.; Franz, B.; Maier, M.; Landthaler, M.; Szeimies, R.-M.; Bäumler, W.

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The role of singlet oxygen and oxygen concentration in photodynamic inactivation of

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bacteria. Proc. Natl. Acad. Sci. USA. 2007, 104, 7223–7228.

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[44] Sies, H. Damage to plasmid DNA by singlet oxygen and its protection. Mutat. Res. 1993, 299, 183-191.



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defects or pre-pores. Biochim. Biophys. Acta, 2011, 1808, 2252 – 2257.

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membrane-active mechanism of a potential α-helical antimicrobial derived from cathelicidin

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PMAP-36. PLoS One. 2014, 9, e86364.

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M. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane.

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Appl. Environ. Microbiol. 2000, 66, 2001–2005.

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curcumin I is associated with damaging of bacterial membrane. PLoS One. 2015, 10,

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Potassium/proton antiport system of Escherichia coli. J. Biol. Chem. 2006, 281, 19822-

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involvement of dihydrofructosazine in the DNA breaking activity of D-glucosamine. Biol.

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[52] Hooper, D. C. Mechanisms of action of antimicrobials: focus on fluoroquinolones. Clin. Infect. Dis. 2001, 32, S9–S15.


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662

Figure Captions

663

Figure 1. Chemical

664

tetrahydroxybutyl)pyrazine (fructosazine) and 2, riboflavin.

structures of the investigated

products, 1,

2,5-bis(D-arabino-

665 666

Figure 2. (A) Optical density curves indicating bacterial growth of E. coli AW 1.7 treated with control (pH 7),

667

acetic acid (pH 5),

1 (pH 7),

1–acetic acid (pH 5),

2 (pH 7)

2-acetic acid (pH 5); (B) Development of SOSG fluorescence as an indicator of 1O2

668

and

669

formation by

670

(C) Intracellular content of 1O2 of cells treated with

671

(pH 7),

672

panel A were determined by non-linear regression. Results are reported as the mean ± SD (n =

673

3). Intracellular 1O2 content (panel C) within each time point is significantly different when

674

letters are different (p < 0.05). When SDs bars are not visible, they were too small to be

675

discernible. NS., not significant.

1 (pH 7),

1-acetic acid (pH 5),

1-acetic acid (pH 5), 2 (pH 7) and

2 (pH 7) and control (pH 7),

2-acetic acid (pH 5; acetic acid (pH 5),

1

2-acetic acid (pH 5). Curve fit lines shown in

676 677

Figure 3. (A) Permeability E. coli’s AW 1.7 outer membrane determined by percentage of 1-N-

678

phenyl-naphthylamine (NPN) uptake. (B) Membrane permeabilization of E. coli AW 1.7

679

measured by percentage of PI uptake as quantitated from flow cytometry data. Treatments in

680

panels A and B are symbolised as follows:

681

1-acetic acid (pH 5), 2 (pH 7) and

control (pH 7),

acetic acid (pH 5),

1 (pH 7),

2-acetic acid (pH 5). Results are reported as the mean ±

682

SD (n = 3). Different letters within each time point represent significant difference (p < 0.05).

683

When SDs bars are not visible, they were too small to be discernible.

684



Page 32 of 42

685

Figure 4. Analyses of fructosazine uptake by E. coli AW 1.7 treated with control (pH 7), acetic

686

acid (pH 5), 1 (pH 7) and 1-acetic acid (pH 5) and collected over time: (A) Representative

687

UHPLC chromatographs obtained from E. coli AW 1.7 treated with 1 at pH 5 and collected over

688

time; (B) MS/MS spectrum of FR peak identified as potassium adduct at m/z 359.4; (C) MS/MS

689

spectrum of 1 at m/z 321.7 and (D) percentage of 1 uptake at

690

represent means ± SD (n = 3). Compound 1 uptake (%) is significantly different when letters are

691

different (* p < 0.05, ** p < 0.01, *** p < 0.001; post hoc Tukey’s test). NS., not significant.

pH 5 and

pH 7. The values

692 693

Figure 5. (A) Fluorescence micrographs showing extent of DNA fragmentation of E. coli AW

694

1.7 and (B) Digital image analysis of nucleoids from E. coli AW 1.7 treated with (a) control, (b)

695

acetic acid (pH 5), (c) 1 (pH 7), (d) 1-acetic acid (pH 5), (e) 2 (pH 7) and (f) 2-acetic acid (pH 5).

696

Bacteria were stained by SYBR Gold stain and liberation of micro granular fibrils from

697

nucleoids was observed under 100× magnification. Representative micrographs of three

698

individual experiments for each treatment are shown. For digital image analysis 60 individual

699

nucleoids from three replications were examined.

700 701

Figure 6. (A) Scanning and (B) transmission electron microscopic images of E. coli AW 1.7

702

treated with control (pH 7), acetic acid (pH 5), 1 (pH 7) and 1–acetic acid (pH 5) for 16 h. Scale

703

bar: 2 µm for SEM and 500 nm for TEM. Representative images of three individual experiments

704

for each treatment are shown.

705 706 707


Page 33 of 42


Figure graphics

Figure 1.

OH

OH

N

OH

OH

1

OH HO

N OH

OH O

H3C

N

H3C

N

NH N

O

2

HO

HO

OH OH



Page 34 of 42

Figure 2.

Fluorescence intensity (AU)

A

B

C


Page 35 of 42


Figure 3. A

B



Page 36 of 42

Figure 4.

1h 2h

A

3h 4h 5h 6h 8h 10 h 12 h 14 h 16 h 0h Standard


Page 37 of 42


Figure 4. B

[M+K]+ [K]+

[M-H2O+H]+ [M+H]



Page 38 of 42

Figure 4. C

[M-H2O+H]+ [M-2H2O+H]+

[M+H]


Page 39 of 42


Figure 4.

** ***

*

***

D

NS ***

** *

NS NS



Page 40 of 42

Figure 5.

A a

b

c

d

e

f

Surface area

B


Page 41 of 42


Figure 6. A

Control (pH 7)

Acetic acid (pH 5)

1 (pH 7)

1-acetic acid (pH 5)

B



Combined effect of fructosazine and acetic acid treatment against heat-resistant E. coli AW 1.7. 85x47mm (300 x 300 DPI)

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