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Neglected antibacterial activity of ethylene glycol as a common solvent
Accepted Manuscript Neglected antibacterial activity of ethylene glycol as a common solvent M. Moghayedi, H. Ahmadzadeh, K. Ghazvini, E.K. Goharshadi PII:
S0882-4010(16)30004-3
DOI:
10.1016/j.micpath.2017.04.022
Reference:
YMPAT 2226
To appear in:
Microbial Pathogenesis
Received Date: 3 January 2016 Revised Date:
6 March 2017
Accepted Date: 19 April 2017
Please cite this article as: Moghayedi M, Ahmadzadeh H, Ghazvini K, Goharshadi EK, Neglected antibacterial activity of ethylene glycol as a common solvent, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.04.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Neglected antibacterial activity of ethylene glycol as a common solvent
M. Moghayedi,a H. Ahmadzadeh,b K. Ghazvini,c E. K. Goharshadib,d* a
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Department of Chemistry, Ferdowsi University of Mashhad, International Campus, Mashhad, Iran b
Department of Chemistry, Ferdowsi University of Mashhad, Mashhad 9177948974, Iran
c
Antimicrobial resistance research center, Department of Microbiology, School of Medicine, Mashhad University of Medical, Iran
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Center of Nano Research, Ferdowsi University of Mashhad, Mashhad 9177948974, Iran
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c
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Corresponding author. E-mail address: gohari@ um.ac.ir (E. K. Goharshadi) Tel: 098-5138805558 Fax: 098-5138795457
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Abstract
For the first time, the antibacterial activity of ethylene glycol (EG), a routine frequently used
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solvent against Escherichia coli (E .coli) bacterium, was assessed. The antibacterial activity of EG against E. coli was measured using colony counting and broth turbidity assays. The influence of EG concentration (1.5 to 25.0%v/v) and exposure time on the growth of E .coli was
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investigated. By increasing EG concentration, its antibacterial activity against E. coli increased so that for both 24.0% and 25.0% of EG, the bacteria growth was totally inhibited within 4 h.
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The MIC and MBC values of EG are 18.0 and 25%v/v, respectively. Since the ratio of MBC to MIC is less than four, EG acts as a bactericidal agent. Also, a model for the slopes of the linear part of the growth curves was proposed. The SEM images of bacteria cells before and after
Keywords
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exposure to EG show that most E .coli were seriously distorted.
Antibacterial activity; ethylene glycol; minimum inhibitory concentration; minimum bactericidal
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concentration; Escherichia coli
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1. Introduction Antibacterial activity is the ability of a compound to kill bacterium or inhibit its growth. The compounds with such properties are called antibacterial agents [1] which are used tocontrol
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different bacterial diseases [2, 3]. With increasing the number of infectious diseases over the world, research for finding novel antibacterial agents is a common practice [4]. For investigating the antibacterial activity of a substance in vitro, various techniques such as broth dilution, agar
SC
dilution, and the microtiter plate-based method have been used [5]. A variety of factors such as nature and structure of antibacterial substances, environmental conditions, and solvents may
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influence the antibacterial activity of an agent [6].
Traditionally, broth dilution technique is frequently applied for measuring the MIC [7]. This technique requires preparing serial dilutions of an antimicrobial agent in a suitable solvent. Among several parameters such as temperature and pH which affect MIC measurements, [8] the
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solvent is the most important one. For the activity assessment of an antibacterial agent, it is essential to selectan appropriate solvent with the least antibacterial activity. Unfortunately, for most of such assessments, the solvent antibacterial activity is not taken into consideration. For
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example, in spite of ethylene glycol (EG) being commonly used as the solvent for dispersing the antibacterial agents in broth media, its antibacterial activity has not been reported yet. Wang et
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al. [9] investigated the antibacterial activity of TiO2 nanoparticles (NPs) cotton fabrics dispersed in EG. They did not measure the antibacterial activity of EG itself. Of course, the antibacterial activity of several different EG-based compounds such as phenyl ether of ethylene glycol [10], propylene glycol, trimethylene glycol ethers [11], and polyethylene glycol 400 [12], were investigated but there is no report in the literature on antibacterial activity of EG by itself.
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The main aim of the present study is to assess the antibacterial activity of EG against Escherichia coli (E .coli.) bacterium. For this purpose, the influence of several EG concentrations (1.5 to 25.0%v/v) and exposure times were investigated on the growth of E .coli.
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Also, a linear model for growth inhibition curves was proposed. E .coli is the gram-negative bacterium which is used in most antibacterial assays as an indicator bacterium and also is the cause of some diseases such as urinary tract infection, diarrhea, hemorrhagic colitis, and
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hemolytic-uremic syndrome [3, 6]. In general, gram-negative bacteria are more resistant to antibacterial agents compared with gram-positive [1]. To elucidate the antibacterial mechanism
EG were recorded.
2. Materials and methods
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2.1.Materials
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of EG, the scanning electron microscopy (SEM) images of bacteria before and after exposure to
Ethylene glycol (99%, Merck, Germany) was used as a solvent to study the growth of E .coli
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Infusion (BHI)).
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(ATCC25922) bacterium and compared with the conventional growth medium (Brain Heart
2.2. Antibacterial assay
The antibacterial activity of EG against E. coli was assayed using broth turbidimetry as a qualitative measure of cell growth and also by colony counting to investigate cell viability [8]. In this study, different concentrations of EG (1.5 to 25.0 v/v) were prepared in BHI broth in various test tubes. Then, 100 µL of freshly grown bacterial inoculums (106 CFU/mL) prepared from overnight growth of bacteria were added to each test tube. E. coli was incubated with different
4
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concentrations (1.5-25.0 v/v %) of EG in broth media. In order to examine the bacterial cell growth, 200 µL of each tube was distributed in three wells in sterile 96-well plates and incubated at 37 oC in a shaking incubator. Using an ELISA plate reader (Stat Fax-2100), , the growth of E.
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coli in a liquid growth medium was investigated by measuring the optical density (OD) at 2, 4, 6, 8, 20, 22, and 24 hours at 630 nm . The same liquid growth medium without microorganisms containing the same concentration of EG was cultured and considered as a blank control. Net
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turbidity of bacterial growth in each concentrations of EG was calculated by distracting the turbidity of the corresponding blank from the test tubescontaining EG. Also, each sample was
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cultured for colony counting on the solid growth medium. The aliquots from each concentrationsof EG were sampled, plated on nutrient agar, and incubated at 37 °C for 24 h. Then, the colonies were counted and recorded as colony forming units (CFU). The death rate of bacteria cells was determined from the growth curve by micro-dilution and colony count
2.3. SEM preparation
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methods.
The morphology of E. coli after treatment with EG was investigated by SEM. For this
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purpose, E. coli (108 CFU/mL) was cultured in a Broth culture medium containing 15% and 25 % of EG for 6 h at 37 o C in a shaking incubator. The suspension was centrifuged for 5 min. The
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bacteria were collected and washed twice with phosphate-buffered saline and then immerged in a beaker containing double-distilled water. The suspension was filtered through a polycarbonate filter (Whatman Nucleopore 0.1 µm) and fixed in a glutaraldehyde solution (3 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate/hydrochloric acid buffer, pH 7.5) at 4 °C for 2 h. The filters containing the sample were washed with the sodium cacodylate/hydrochloric acid buffer three times. After several washing with double-distilled water, the sample was dehydrated
5
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successively with different ethanol solutions (50% for 30 min, 75%, 85%, and 95% each for 10 min, and 100% twice for 10 min). Then, the sample was dried to remove ethanol completely. Finally, it was riding onto an aluminum stub, coated by gold sputter, and observed under a
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scanning electron microscope.
3. Results
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3.1. Time and concentration dependence of EG antibacterial activity
The effect of EG concentrations and times on the growth curves of E. coli is illustrated in Fig.
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1. As Fig. 1a and Fig. 1b show the growth of E. coli decreases by increasing EG concentrations and almost is inhibited at approximately 18.0% EG. The OD630 values became zero for EG with concentrations of 18.0, 20.0, 24.0, and 25.0 %v/v implying that the bacterial growth was completely inhibited (the arrow in Fig.1b). Hence, by increasing EG concentrations, the
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antibacterial activity increased. At low concentrations of EG (up to 18%), at first with elapsing time the OD values increased slowly and then raised sharply. Finally, it approached to a plateau or decreased because of the absence of the necessary nutrients or production of toxic materials.
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As Table 1 shows the percentage of growth inhibition (GI %) of E. coli rises by increasing EG concentrations. Of course, in the range of 1.5- 3.0%, EG has no effect on the bacterial growth.
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Only a slight reduction in the growth rate of E. coli was observed for 6.0 % EG. For concentrations in the range of 10.0- 12.0% EG, the growth inhibition increased until 6 h and then decreased. The growth inhibition value increased for 15.0% EG up to 8 h and then it decreased with time. The growth inhibition increased with time for 18.0 and 20.0% EG. For both 24.0% and 25.0% EG, the bacteria growth wasinhibited totally within 4 h. After this time, no change was observed.
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To more accurately investigatethe EG antibacterial activity, we also used colony count method. Fig. 2 shows the number of viable cells of bacteria at different times for various concentrations of EG . As this Figure shows the number of viable bacteria reduces with
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increasing the EG concentration.
As Fig. 2 shows a significant decrease in the population of viable cells is observed when EG is included in the growth medium. At least, 2-log reduction in viability for 10% to 25 % EG
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compared with that of the control experiment is observed. There was no viable bacterium at concentration of 25.0% EG. The bacterial growth inhibition from colony counting data matches
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well with the results obtained from OD when Figures 1 and 2 are compared.
3.2. Bactericidal and bacteriostatic determination of EG concentrations The MIC and MBC values were determined by OD measurement and colony count methods,
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respectively. The MIC and MBC values are 18.0%v/v and 25%v/v EG, respectively.
4. Discussion
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4.1. Antibacterial activity of EG
The antibacterial activity of EG against E .coli. as functions of both EG concentrations and
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exposure times was assessed. Generally, the growth of bacteria contains four phases: lag, exponential growth, stationary, and death phases [13]. To gain further insight into antibacterial activity of EG, the slopes of the linear part of growth phase (Fig. 1) against EG concentrations were plotted and shown in Fig. 3. With increasing the EG concentrations, the slopes decreased exponentially and became zero for 18 to 25% EG. The plot was fitted by the following equation with the R2 value of 0.98: =
+
(1) 7
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where So, a, and b are -0.03, 0.28, and 0.11, respectively. S and C stand for the slope of linear part of growth phase and the concentration of EG, respectively.
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The ratio of MBC to MIC can be used for determining the bacteriostatic and bactericidal agents in vitro. If the ratio of MBC to MIC is greater than 4, the agent is said to be bacteriostatic. Otherwise, it is called a bactericidal agent [14]. Since, the ratio of MBC to MIC is less than four,
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EG acts as a bactericidal agent. Of course, it should be mentioned that different parameters such as growth conditions, bacterial density, and test time can change the values of MIC and MBC.
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4.2. The effect of EG on bacterial membrane morphology changes
To further understand the antibacterial mechanism of EG, the morphology changes and integrity of cell membranes of bacteria were visualized evaluated using SEM (LEO 1450 VP model). Fig. 4 shows the SEM images of bacteria cells before and after treated with EG. With no
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exposure to EG. E. coli membranes are intact and smooth (Figs. 4a,4b). When exposed to different concentrations of EG, some E. coli bacteria were deformed under exposure to 15% EG (Figs. 4c,4d). Most E. coli were damaged when treated with 25% EG (Figs. 4e and 4f). Some
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cells were elongated and some show pore formation on their surfaces. This may promote the entrance of reactive species into the cell and facilitate the bactericidal effect. On the other hand,
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the cell shape was seriously distorted and the cell membrane was greatly ruptured which leads to cytoplasm leakage and cell death.
5. Conclusion For the first time, this work assessed the antibacterial activity of a frequently used solvent, i.e. EG. The main conclusions are: 8
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1. By increasing EG concentrations, its antibacterial activity against E .coli increased so that for both 24.0% and 25.0% EG, the bacteria growth was totally inhibited within 4 h. 2. The number of viable bacteria reduced with increasing the concentrations of EG. At least,
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2-log reduction in viability for 10% to 25 % EG was observed compared with those of the control experiment. There was no viable bacterium for 25.0% EG.
3. The MIC and MBC values were 18.0 and 25%v/v EG, respectively. Since, the ratio of
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MBC to MIC is less than four, EG acted as a bactericidal agent.
4. With increasing the EG concentrations, the slopes of the linear part of growth phase
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against EG concentration decreased exponentially and became zero for 18 to 25% EG. 5. The SEM images of bacteria cells before and after exposure showed most E .coli were damaged.
We suggest the antibacterial activity of solvent should be taken into consideration in evaluating
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the antibacterial activity of an agent.
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Acknowledgment
The authors acknowledge Ferdowsi University of Mashhad, International Campus for supporting
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this project (3/33463). We also thank antimicrobial resistance research center of Mashhad University of Medical Sciences for technical supports.
Conflict of Interests
No conflict of interest declared.
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References [1] M.J. Hajipour, K.M. Fromm, A. Ashkarran, D.J. Aberasturi, I.R. Larramendi, , T. Rojo, V. Serpooshan, W.J. Parak, and M. Mahmoudi, Antibacterial properties of nanoparticles. Trends
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Bio. 30 (2012) 499-511.
[2] N. Jones, B. Ray, K. Ranjit, and A. Manna, Antibacterial activity of ZnO nanoparticle
suspensions on a broad spectrum of microorganisms. FEMS Microbiol Lett. 279 (2008) 71–76.
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[3] M. Zarei, A. Jamnejad, and E. Khajehali, Antibacterial effect of silver nanoparticles against four foodborne pathogens. Jundishapur J Microbiol. 7(2014) 1-4.
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[4] R. Jalal, E.K. Goharshadi, M. Abareshi, M. Moosavi, and P. Nancarrow, ZnO nanofluids: Green synthesis, characterization, and antibacterial activity. Mater Chem Phys. 121 (2010) 198–201.
[5] A. Sirelkhatim, S. Mahmud, A. Seeni, N.H.M. Kaus, L.C. Ann, S.K.M. Bakhori, H. Hasan,
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and D. Mohamad, Review on Zinc Oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Lett. 7(2015) 219–242. [6] P.G. Mazzola, A.F. Jozala, L.C. L. Novaes, P. Moriel, and T.C.V. Penna, Minimal inhibitory
(2009) 241-248.
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concentration (MIC) determination of disinfectant and/or sterilizing agents. Braz J Pharm Sci. 45
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[7] R.J.W. Lambert, P.N. Skandamis, P.J. Coote, and G. J.E. Nychas, A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol, and carvacrol. J Appl Microbiol. 91(2001) 453-462. [8] R. Schwalbe, L. Steele-Moore, A.C. Goodwin, (2007) Antimicrobial susceptibility testing protocols, pp.75-79, New York: CRC Press. [9] L.M. Wang, Y. Shen, H.F. Zhang, J.C. Wang, Y. Ding, and W.T. Qing,
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Effect of nano-TiO2 particles surface modification on antibacterial properties of cotton fabrics. JFBI. 3 (2010) 224-230.
247 (1944) 175-176.
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[10] H. Berry, Antibacterial values of ethylene glycol monophenyl ether (Phenoxetol). Lancet.
[11] F. M. Berger, C. V. Hubbard, and B. J. Ludwig, The antimicrobial action of certain glycerol ethers and related compounds. Appl. Microbiol. 1 (1953) 146–149.
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[12] J. Chirife, L. Herszage, A. Joseph, J.P. Bozzini, N. Leardin, and Kohn, E.S. In vitro
Chemother. 24 (1983) 409-412.
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antibacterial activity of concentrated polyethylene glycol 400 solutions. Antimicrob Agents
[13] S.P. Borriello, P.R. Murray, and G. Funke, (2005) Topley and Wilson's, microbiology & microbial infections, Bacteriology. Vol. 1, 10th ed., pp. 39, London: Hodder Education Publisher.
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[14] G.A. Pankey, and L.D. Sabath, Clinical relevance of bacteriostatic versus bactericidal
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870.
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mechanisms of action in the treatment of Gram-Positive bacterial infections. CID. 38(2004) 864-
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Table 1. Percent GI a after exposure to EG with various concentrations and different times at 37
Time (h)
a
Concentration (v/v% ) 1.5
3.0 6.0 10.0 12.0 15.0
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ºC
18.0 20.0
24.0
25.0
2.3
0.0 3.5 38.6 85.3 93.8
93.3 82.6 100.0 100.0
6
2.9
3.1 9.6 78.6 88.9 98.7
99.3 97.8 100.0 100.0
8
0.0
0.0 6.1 52.4 74.4 98.6
99.5 98.6 100.0 100.0
20
0.0
0.0 8.5 24.9 39.2 82.4 100.0 99.1 100.0 100.0
22
0.0
0.0 9.3 24.8 41.2 70.1
99.9 99.2 100.0 100.0
24
0.0
0.0 9.3 26.7 43.4 69.9
99.9 99.3 100.0 100.0
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4
12
% = (1 −
100
(
(
×
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Figure Captions Fig.1. The growth curves of E. coli cells (106 CFU/ ml) exposed to EG with concentrations of (a) 1.5-10.0 % (b) 15.0- 25.0%.
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Fig. 2. E. coli viable cells remaining after exposure to EG over the course of 24 h. Fig. 3. The slopes of the liner part of the growth curve of E. coli against EG concentration.
Fig. 4. SEM images of E. Coli (a) and (b) control with two magnifications (c) and (d) exposure
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with 15% of EG with two magnifications (e) and (f) treated with 25% of EG with different
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magnifications.
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Fig. 1
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Fig. 2
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Fig. 3.
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Fig. 4.
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ACCEPTED MANUSCRIPT The antibacterial activity of ethylene glycol (EG) against E. coli was measured. The MIC and MBC values of EG were measured as 18.0 and 25%v/v, respectively.
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Since the ratio of MIC to MBC is less than four, EG acts as a bactericidal agent.
S0882-4010(16)30004-3
DOI:
10.1016/j.micpath.2017.04.022
Reference:
YMPAT 2226
To appear in:
Microbial Pathogenesis
Received Date: 3 January 2016 Revised Date:
6 March 2017
Accepted Date: 19 April 2017
Please cite this article as: Moghayedi M, Ahmadzadeh H, Ghazvini K, Goharshadi EK, Neglected antibacterial activity of ethylene glycol as a common solvent, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.04.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
Neglected antibacterial activity of ethylene glycol as a common solvent
M. Moghayedi,a H. Ahmadzadeh,b K. Ghazvini,c E. K. Goharshadib,d* a
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Department of Chemistry, Ferdowsi University of Mashhad, International Campus, Mashhad, Iran b
Department of Chemistry, Ferdowsi University of Mashhad, Mashhad 9177948974, Iran
c
Antimicrobial resistance research center, Department of Microbiology, School of Medicine, Mashhad University of Medical, Iran
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Center of Nano Research, Ferdowsi University of Mashhad, Mashhad 9177948974, Iran
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c
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Corresponding author. E-mail address: gohari@ um.ac.ir (E. K. Goharshadi) Tel: 098-5138805558 Fax: 098-5138795457
1
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Abstract
For the first time, the antibacterial activity of ethylene glycol (EG), a routine frequently used
RI PT
solvent against Escherichia coli (E .coli) bacterium, was assessed. The antibacterial activity of EG against E. coli was measured using colony counting and broth turbidity assays. The influence of EG concentration (1.5 to 25.0%v/v) and exposure time on the growth of E .coli was
SC
investigated. By increasing EG concentration, its antibacterial activity against E. coli increased so that for both 24.0% and 25.0% of EG, the bacteria growth was totally inhibited within 4 h.
M AN U
The MIC and MBC values of EG are 18.0 and 25%v/v, respectively. Since the ratio of MBC to MIC is less than four, EG acts as a bactericidal agent. Also, a model for the slopes of the linear part of the growth curves was proposed. The SEM images of bacteria cells before and after
Keywords
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exposure to EG show that most E .coli were seriously distorted.
Antibacterial activity; ethylene glycol; minimum inhibitory concentration; minimum bactericidal
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EP
concentration; Escherichia coli
2
ACCEPTED MANUSCRIPT
1. Introduction Antibacterial activity is the ability of a compound to kill bacterium or inhibit its growth. The compounds with such properties are called antibacterial agents [1] which are used tocontrol
RI PT
different bacterial diseases [2, 3]. With increasing the number of infectious diseases over the world, research for finding novel antibacterial agents is a common practice [4]. For investigating the antibacterial activity of a substance in vitro, various techniques such as broth dilution, agar
SC
dilution, and the microtiter plate-based method have been used [5]. A variety of factors such as nature and structure of antibacterial substances, environmental conditions, and solvents may
M AN U
influence the antibacterial activity of an agent [6].
Traditionally, broth dilution technique is frequently applied for measuring the MIC [7]. This technique requires preparing serial dilutions of an antimicrobial agent in a suitable solvent. Among several parameters such as temperature and pH which affect MIC measurements, [8] the
TE D
solvent is the most important one. For the activity assessment of an antibacterial agent, it is essential to selectan appropriate solvent with the least antibacterial activity. Unfortunately, for most of such assessments, the solvent antibacterial activity is not taken into consideration. For
EP
example, in spite of ethylene glycol (EG) being commonly used as the solvent for dispersing the antibacterial agents in broth media, its antibacterial activity has not been reported yet. Wang et
AC C
al. [9] investigated the antibacterial activity of TiO2 nanoparticles (NPs) cotton fabrics dispersed in EG. They did not measure the antibacterial activity of EG itself. Of course, the antibacterial activity of several different EG-based compounds such as phenyl ether of ethylene glycol [10], propylene glycol, trimethylene glycol ethers [11], and polyethylene glycol 400 [12], were investigated but there is no report in the literature on antibacterial activity of EG by itself.
3
ACCEPTED MANUSCRIPT
The main aim of the present study is to assess the antibacterial activity of EG against Escherichia coli (E .coli.) bacterium. For this purpose, the influence of several EG concentrations (1.5 to 25.0%v/v) and exposure times were investigated on the growth of E .coli.
RI PT
Also, a linear model for growth inhibition curves was proposed. E .coli is the gram-negative bacterium which is used in most antibacterial assays as an indicator bacterium and also is the cause of some diseases such as urinary tract infection, diarrhea, hemorrhagic colitis, and
SC
hemolytic-uremic syndrome [3, 6]. In general, gram-negative bacteria are more resistant to antibacterial agents compared with gram-positive [1]. To elucidate the antibacterial mechanism
EG were recorded.
2. Materials and methods
TE D
2.1.Materials
M AN U
of EG, the scanning electron microscopy (SEM) images of bacteria before and after exposure to
Ethylene glycol (99%, Merck, Germany) was used as a solvent to study the growth of E .coli
AC C
Infusion (BHI)).
EP
(ATCC25922) bacterium and compared with the conventional growth medium (Brain Heart
2.2. Antibacterial assay
The antibacterial activity of EG against E. coli was assayed using broth turbidimetry as a qualitative measure of cell growth and also by colony counting to investigate cell viability [8]. In this study, different concentrations of EG (1.5 to 25.0 v/v) were prepared in BHI broth in various test tubes. Then, 100 µL of freshly grown bacterial inoculums (106 CFU/mL) prepared from overnight growth of bacteria were added to each test tube. E. coli was incubated with different
4
ACCEPTED MANUSCRIPT
concentrations (1.5-25.0 v/v %) of EG in broth media. In order to examine the bacterial cell growth, 200 µL of each tube was distributed in three wells in sterile 96-well plates and incubated at 37 oC in a shaking incubator. Using an ELISA plate reader (Stat Fax-2100), , the growth of E.
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coli in a liquid growth medium was investigated by measuring the optical density (OD) at 2, 4, 6, 8, 20, 22, and 24 hours at 630 nm . The same liquid growth medium without microorganisms containing the same concentration of EG was cultured and considered as a blank control. Net
SC
turbidity of bacterial growth in each concentrations of EG was calculated by distracting the turbidity of the corresponding blank from the test tubescontaining EG. Also, each sample was
M AN U
cultured for colony counting on the solid growth medium. The aliquots from each concentrationsof EG were sampled, plated on nutrient agar, and incubated at 37 °C for 24 h. Then, the colonies were counted and recorded as colony forming units (CFU). The death rate of bacteria cells was determined from the growth curve by micro-dilution and colony count
2.3. SEM preparation
TE D
methods.
The morphology of E. coli after treatment with EG was investigated by SEM. For this
EP
purpose, E. coli (108 CFU/mL) was cultured in a Broth culture medium containing 15% and 25 % of EG for 6 h at 37 o C in a shaking incubator. The suspension was centrifuged for 5 min. The
AC C
bacteria were collected and washed twice with phosphate-buffered saline and then immerged in a beaker containing double-distilled water. The suspension was filtered through a polycarbonate filter (Whatman Nucleopore 0.1 µm) and fixed in a glutaraldehyde solution (3 mL of 2.5% glutaraldehyde in 0.1 M sodium cacodylate/hydrochloric acid buffer, pH 7.5) at 4 °C for 2 h. The filters containing the sample were washed with the sodium cacodylate/hydrochloric acid buffer three times. After several washing with double-distilled water, the sample was dehydrated
5
ACCEPTED MANUSCRIPT
successively with different ethanol solutions (50% for 30 min, 75%, 85%, and 95% each for 10 min, and 100% twice for 10 min). Then, the sample was dried to remove ethanol completely. Finally, it was riding onto an aluminum stub, coated by gold sputter, and observed under a
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scanning electron microscope.
3. Results
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3.1. Time and concentration dependence of EG antibacterial activity
The effect of EG concentrations and times on the growth curves of E. coli is illustrated in Fig.
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1. As Fig. 1a and Fig. 1b show the growth of E. coli decreases by increasing EG concentrations and almost is inhibited at approximately 18.0% EG. The OD630 values became zero for EG with concentrations of 18.0, 20.0, 24.0, and 25.0 %v/v implying that the bacterial growth was completely inhibited (the arrow in Fig.1b). Hence, by increasing EG concentrations, the
TE D
antibacterial activity increased. At low concentrations of EG (up to 18%), at first with elapsing time the OD values increased slowly and then raised sharply. Finally, it approached to a plateau or decreased because of the absence of the necessary nutrients or production of toxic materials.
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As Table 1 shows the percentage of growth inhibition (GI %) of E. coli rises by increasing EG concentrations. Of course, in the range of 1.5- 3.0%, EG has no effect on the bacterial growth.
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Only a slight reduction in the growth rate of E. coli was observed for 6.0 % EG. For concentrations in the range of 10.0- 12.0% EG, the growth inhibition increased until 6 h and then decreased. The growth inhibition value increased for 15.0% EG up to 8 h and then it decreased with time. The growth inhibition increased with time for 18.0 and 20.0% EG. For both 24.0% and 25.0% EG, the bacteria growth wasinhibited totally within 4 h. After this time, no change was observed.
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To more accurately investigatethe EG antibacterial activity, we also used colony count method. Fig. 2 shows the number of viable cells of bacteria at different times for various concentrations of EG . As this Figure shows the number of viable bacteria reduces with
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increasing the EG concentration.
As Fig. 2 shows a significant decrease in the population of viable cells is observed when EG is included in the growth medium. At least, 2-log reduction in viability for 10% to 25 % EG
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compared with that of the control experiment is observed. There was no viable bacterium at concentration of 25.0% EG. The bacterial growth inhibition from colony counting data matches
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well with the results obtained from OD when Figures 1 and 2 are compared.
3.2. Bactericidal and bacteriostatic determination of EG concentrations The MIC and MBC values were determined by OD measurement and colony count methods,
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respectively. The MIC and MBC values are 18.0%v/v and 25%v/v EG, respectively.
4. Discussion
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4.1. Antibacterial activity of EG
The antibacterial activity of EG against E .coli. as functions of both EG concentrations and
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exposure times was assessed. Generally, the growth of bacteria contains four phases: lag, exponential growth, stationary, and death phases [13]. To gain further insight into antibacterial activity of EG, the slopes of the linear part of growth phase (Fig. 1) against EG concentrations were plotted and shown in Fig. 3. With increasing the EG concentrations, the slopes decreased exponentially and became zero for 18 to 25% EG. The plot was fitted by the following equation with the R2 value of 0.98: =
+
(1) 7
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where So, a, and b are -0.03, 0.28, and 0.11, respectively. S and C stand for the slope of linear part of growth phase and the concentration of EG, respectively.
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The ratio of MBC to MIC can be used for determining the bacteriostatic and bactericidal agents in vitro. If the ratio of MBC to MIC is greater than 4, the agent is said to be bacteriostatic. Otherwise, it is called a bactericidal agent [14]. Since, the ratio of MBC to MIC is less than four,
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EG acts as a bactericidal agent. Of course, it should be mentioned that different parameters such as growth conditions, bacterial density, and test time can change the values of MIC and MBC.
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4.2. The effect of EG on bacterial membrane morphology changes
To further understand the antibacterial mechanism of EG, the morphology changes and integrity of cell membranes of bacteria were visualized evaluated using SEM (LEO 1450 VP model). Fig. 4 shows the SEM images of bacteria cells before and after treated with EG. With no
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exposure to EG. E. coli membranes are intact and smooth (Figs. 4a,4b). When exposed to different concentrations of EG, some E. coli bacteria were deformed under exposure to 15% EG (Figs. 4c,4d). Most E. coli were damaged when treated with 25% EG (Figs. 4e and 4f). Some
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cells were elongated and some show pore formation on their surfaces. This may promote the entrance of reactive species into the cell and facilitate the bactericidal effect. On the other hand,
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the cell shape was seriously distorted and the cell membrane was greatly ruptured which leads to cytoplasm leakage and cell death.
5. Conclusion For the first time, this work assessed the antibacterial activity of a frequently used solvent, i.e. EG. The main conclusions are: 8
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1. By increasing EG concentrations, its antibacterial activity against E .coli increased so that for both 24.0% and 25.0% EG, the bacteria growth was totally inhibited within 4 h. 2. The number of viable bacteria reduced with increasing the concentrations of EG. At least,
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2-log reduction in viability for 10% to 25 % EG was observed compared with those of the control experiment. There was no viable bacterium for 25.0% EG.
3. The MIC and MBC values were 18.0 and 25%v/v EG, respectively. Since, the ratio of
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MBC to MIC is less than four, EG acted as a bactericidal agent.
4. With increasing the EG concentrations, the slopes of the linear part of growth phase
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against EG concentration decreased exponentially and became zero for 18 to 25% EG. 5. The SEM images of bacteria cells before and after exposure showed most E .coli were damaged.
We suggest the antibacterial activity of solvent should be taken into consideration in evaluating
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the antibacterial activity of an agent.
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Acknowledgment
The authors acknowledge Ferdowsi University of Mashhad, International Campus for supporting
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this project (3/33463). We also thank antimicrobial resistance research center of Mashhad University of Medical Sciences for technical supports.
Conflict of Interests
No conflict of interest declared.
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Bio. 30 (2012) 499-511.
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[3] M. Zarei, A. Jamnejad, and E. Khajehali, Antibacterial effect of silver nanoparticles against four foodborne pathogens. Jundishapur J Microbiol. 7(2014) 1-4.
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and D. Mohamad, Review on Zinc Oxide nanoparticles: Antibacterial activity and toxicity mechanism. Nano-Micro Lett. 7(2015) 219–242. [6] P.G. Mazzola, A.F. Jozala, L.C. L. Novaes, P. Moriel, and T.C.V. Penna, Minimal inhibitory
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concentration (MIC) determination of disinfectant and/or sterilizing agents. Braz J Pharm Sci. 45
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[7] R.J.W. Lambert, P.N. Skandamis, P.J. Coote, and G. J.E. Nychas, A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol, and carvacrol. J Appl Microbiol. 91(2001) 453-462. [8] R. Schwalbe, L. Steele-Moore, A.C. Goodwin, (2007) Antimicrobial susceptibility testing protocols, pp.75-79, New York: CRC Press. [9] L.M. Wang, Y. Shen, H.F. Zhang, J.C. Wang, Y. Ding, and W.T. Qing,
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[10] H. Berry, Antibacterial values of ethylene glycol monophenyl ether (Phenoxetol). Lancet.
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[12] J. Chirife, L. Herszage, A. Joseph, J.P. Bozzini, N. Leardin, and Kohn, E.S. In vitro
Chemother. 24 (1983) 409-412.
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antibacterial activity of concentrated polyethylene glycol 400 solutions. Antimicrob Agents
[13] S.P. Borriello, P.R. Murray, and G. Funke, (2005) Topley and Wilson's, microbiology & microbial infections, Bacteriology. Vol. 1, 10th ed., pp. 39, London: Hodder Education Publisher.
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[14] G.A. Pankey, and L.D. Sabath, Clinical relevance of bacteriostatic versus bactericidal
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870.
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mechanisms of action in the treatment of Gram-Positive bacterial infections. CID. 38(2004) 864-
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Table 1. Percent GI a after exposure to EG with various concentrations and different times at 37
Time (h)
a
Concentration (v/v% ) 1.5
3.0 6.0 10.0 12.0 15.0
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ºC
18.0 20.0
24.0
25.0
2.3
0.0 3.5 38.6 85.3 93.8
93.3 82.6 100.0 100.0
6
2.9
3.1 9.6 78.6 88.9 98.7
99.3 97.8 100.0 100.0
8
0.0
0.0 6.1 52.4 74.4 98.6
99.5 98.6 100.0 100.0
20
0.0
0.0 8.5 24.9 39.2 82.4 100.0 99.1 100.0 100.0
22
0.0
0.0 9.3 24.8 41.2 70.1
99.9 99.2 100.0 100.0
24
0.0
0.0 9.3 26.7 43.4 69.9
99.9 99.3 100.0 100.0
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4
12
% = (1 −
100
(
(
×
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Figure Captions Fig.1. The growth curves of E. coli cells (106 CFU/ ml) exposed to EG with concentrations of (a) 1.5-10.0 % (b) 15.0- 25.0%.
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Fig. 2. E. coli viable cells remaining after exposure to EG over the course of 24 h. Fig. 3. The slopes of the liner part of the growth curve of E. coli against EG concentration.
Fig. 4. SEM images of E. Coli (a) and (b) control with two magnifications (c) and (d) exposure
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with 15% of EG with two magnifications (e) and (f) treated with 25% of EG with different
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magnifications.
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Fig. 1
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Fig. 2
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Fig. 3.
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Fig. 4.
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Since the ratio of MIC to MBC is less than four, EG acts as a bactericidal agent.