In vitro enhancement of antibiotic susceptibility of drug resistant Escherichia coli by cinnamaldehyde

Accepted Manuscript In vitro enhancement of antibiotic susceptibility of drug resistant Escherichia coli by cinnamaldehyde

Jeyachchandran Visvalingam, Kavitha Palaniappan, Richard A. Holley PII:

S0956-7135(17)30196-2

DOI:

10.1016/j.foodcont.2017.04.011

Reference:

JFCO 5563

To appear in:

Food Control

Received Date:

13 December 2016

Revised Date:

06 April 2017

Accepted Date:

08 April 2017

Please cite this article as: Jeyachchandran Visvalingam, Kavitha Palaniappan, Richard A. Holley, In vitro enhancement of antibiotic susceptibility of drug resistant Escherichia coli by cinnamaldehyde, Food Control (2017), doi: 10.1016/j.foodcont.2017.04.011

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Title: In vitro enhancement of antibiotic susceptibility of drug resistant Escherichia coli

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by cinnamaldehyde

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Jeyachchandran Visvalingam1†, Kavitha Palaniappan 1, and Richard A. Holley1*

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Canada.

Department of Food Science, University of Manitoba, Winnipeg, Manitoba, R3T 2N2,

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*Author for Correspondence: Department of Food Science, University of Manitoba,

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Winnipeg, Manitoba R3T N2, Canada; E-Mail: [email protected]; Tel.: +1-519-

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874-1103; Fax: +1-204-474-7630.

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†Current

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Development Centre, 6000 C & E Trail, Lacombe, Alberta T4L 1W1, Canada.

address: Agriculture and Agri-Food Canada, Lacombe Research and

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Abstract

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Cinnamaldehyde is a natural antimicrobial compound that has been found to damage the

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cytoplasmic membrane, inhibit septum development and cause cell elongation, as well as

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induce oxidative stress in Escherichia coli. Thus, cinnamaldehyde may be of value as a

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natural compound to enhance susceptibility of drug resistant E. coli to antibiotics in

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livestock production. This study examined the ability of cinnamaldehyde to increase the

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susceptibility of E. coli to antibiotics using the checkerboard method. Interactions

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between the antimicrobials were characterized using fractional inhibitory concentration

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(FIC) values. All tested E. coli strains were resistant to erythromycin and bacitracin but

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were susceptible to penicillin G and ampicillin. Strains 8WT, ATCC 23739 and 02:0627

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were resistant to novobiocin and the latter two strains were also resistant to tetracycline.

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Cinnamaldehyde synergistically increased the susceptibility of all E. coli strains to

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erythromycin (FIC ≤ 0.5). Another synergistic effect between tetracycline and

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cinnamaldehyde was observed when tested against E. coli ATCC 23739 (FIC = 0.3).

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Cinnamaldehyde synergistically and additively reduced the MIC of novobiocin when

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tested against ATCC 23739 and 02:0627 (FIC ≤ 0.5) or 8WT (FIC = 1), respectively,

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suggesting that E. coli strains may respond differently to challenge by the

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cinnamaldehyde-novobiocin combination. With all strains, cinnamaldehyde was not

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effective at reducing the MIC value of bacitracin. Findings of this study suggest that

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cinnamaldehyde may be used in combination with several antibiotics to enhance

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susceptibility of drug resistant E. coli.

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Key words: Escherichia coli, antibiotic resistance, cinnamaldehyde

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1. Introduction

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Since their discovery, antibiotics have played an irreplaceable role in reducing illnesses

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and deaths associated with infectious diseases in humans and animals. However, selective

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pressure resulting from their use in human medicine and animal production has been

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implicated in the enhanced survival of resistant bacteria as well as the retention,

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accumulation and dispersion of antibiotic resistant genes among diverse groups of

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bacteria (Mathew, Cissell, & Liamthong, 2007; Spellberg , Bartlett , & Gilbert 2013).

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Escherichia coli, a commensal gut microorganism of humans and animals, can spread

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through food, water, and by human to human contact (Ewers, Bethe, Semmler, Guenther,

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& Wieler, 2012; Kuenzli, et al., 2014; Wu, et al., 2013). A significant proportion of E.

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coli isolates, including its pathogenic variants from cattle, other food animals, meat, fish,

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seafood and humans have been found to be resistant to one or more antibiotics and to

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carry transferable resistance genes (Aslam, Diarra, Service, & Rempel, 2009; Aslam,

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Stanford, & McAllister, 2010; Ryu, et al., 2012; Sheikh, et al., 2012; Tadesse, et al.,

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2012).

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In contrast with an increase in the prevalence of antibiotic resistant bacteria in various

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environmental settings and an increase in related infections, the pace of antibiotic

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discovery has been much slower, which in turn may limit the available treatment options

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for resistant bacterial infections (Ferri, Ranucci, Romagnoli, & Giaccone, 2015).

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Therefore, significant effort has been undertaken to evaluate combination of plant

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essential oils and their components like cinnamaldehyde, carvacrol and thymol with

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antibiotics to increase the susceptibility of drug resistant bacteria. Many plant essential

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oils and their components have been found to enhance the activity of some antibiotics

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against drug resistant bacteria (Langeveld, Veldhuizen, & Burt, 2014).

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Cinnamaldehyde, a major component of cinnamon oil, is a very effective antimicrobial

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against many foodborne bacteria, including E. coli (Amalaradjou, et al., 2010; Ayari,

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Dussault, Jerbi, Hamdi, & Lacroix, 2012; Ye, et al., 2013). Unlike antibiotics, which

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mostly disrupt single cellular processes in bacteria (Langeveld, et al., 2014),

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cinnamaldehyde has been shown to disrupt the cytoplasmic membrane and inhibit

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membrane ATPase activity (Gill & Holley, 2006; Visvalingam & Holley, 2012), to

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induce oxidative stress and repress expression of DNA, protein, O-antigen, and fimbrial

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synthetic genes (Visvalingam, Hernandez-Doria, & Holley, 2013). In addition,

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cinnamaldehyde has also been found to bind to the cell division protein, FtsZ which led

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to inhibition of its GTPase activity, septum development, and induction of cell elongation

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(Domadia, Swarup, Bhunia, Sivaraman, & Dasgupta, 2007; Visvalingam & Holley, 2012).

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Similarly, binding of cinnamaldehyde to the tryptophan and tyrosine residues of β-

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galactosidase has led to conformational change and inhibition of its activity in vitro

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(Wang, Wang, Zeng, Gong, & Huang, 2017). These observations suggested that

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cinnamaldehyde should be examined for its ability to interact with antibiotics and

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enhance susceptibility of drug resistant E. coli. However, cinnamaldehyde’s ability to

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inhibit multiple cellular processes, and the extent of these effects appeared to vary among

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E. coli strains (Visvalingam & Holley, 2012), and this may influence the overall

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combined antimicrobial effect of cinnamaldehyde with antibiotics. Therefore, the

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objective of this study was to characterize how cinnamaldehyde might influence the

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antibiotic susceptibility of E. coli strains with different environmental backgrounds.

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2. Materials and methods

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2.1. Bacterial strains and inoculum preparation

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An E. coli isolate 8WT from beef and a human isolate of E. coli O157:H7 (02:0627) that

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were previously used to study the effect of cinnamaldehyde (Visvalingam, et al., 2013;

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Visvalingam & Holley, 2012) and two E. coli strains from the American type culture

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collection, ATCC 11775 and ATCC 23739, were used in this study. Stock cultures of E.

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coli were maintained in Brain Heart Infusion broth (BHIB, Accumedia, Lansing, MI, US)

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containing 15 % glycerol at -80 ºC. Working cultures of E. coli were maintained on Brain

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Heart Infusion agar (BHIA, Oxoid, Fisher Scientific, Edmonton, AB, Canada) at 4 ºC.

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Prior to each experiment, a single colony was transferred to 10 ml BHIB and incubated at

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37 ºC for 18 h. Then 100 µl bacterial suspension was transferred to 9.9 ml BHIB and

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incubated at 37 ºC for 2 to 3 h to obtain log phase cultures. Bacterial density was adjusted

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to 1 x 108 CFU/ml using a spectrophotometer (Ultraspec 2000, Pharmacia Biotech,

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Cambridge, England) and serially diluted to get 1 x 106 CFU/ml.

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2.2. Antimicrobial compounds

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Tetracycline, ampicillin, penicillin G, erythromycin and novobiocin (Sigma-Aldrich

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Canada Ltd, Oakville, ON, Canada) were selected from the list of antibiotics that are

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permitted for use as animal feed additives in Canada. Cinnamaldehyde was obtained from

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Sigma-Aldrich. All antimicrobial compounds were analytical grade products.

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2.3. Minimal inhibitory concentration determination

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Minimal inhibitory concentrations (MIC) of antibiotics were determined using the broth

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microdilution assay described by Wiegand, Hilpert, and Hancock (2008) and CLSI (2002)

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with some modifications. Stock solutions of ampicillin (8 µg/ml), penicillin G (64 µg/ml),

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erythromycin (128 µg/ml), bacitracin (2048 µg/ml) and novobiocin (512 µg/ml) were

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prepared in distilled water. Stock solutions of tetracycline (256 µg/ml) and

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cinnamaldehyde (3200 µg/ml) were prepared in distilled water containing 4 % (V/V)

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dimethyl sulfoxide (DMSO, Sigma-Aldrich). The concentrations of stock solutions were

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chosen based on the MIC values obtained for E. coli and Salmonella Typhimurium SGI 1

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(tet A) in a previous study (Palaniappan & Holley, 2010). All antibiotic solutions were

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prepared and sterilized using 0.2 µm filters (Fisher) before each use. Each well of the 96

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well microplate (Falcon no 3072, Becton Dickinson and Co., Franklin Lakes, NJ, US)

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was filled with 50 µl double-strength BHIB. A 50 µl aliquot of antibiotic or

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cinnamaldehyde stock solution was added to the first well, serially diluted in the

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microwell plate and the content of the last well was discarded. A 50 µl portion of diluted

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bacterial suspension was added to each well to get an initial inoculum of 5 x 105 CFU/ml.

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A set of wells containing similar concentrations of antibiotics or cinnamaldehyde without

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inoculum (negative control), a well containing only inoculum (positive control), and a

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well containing 1% (V/V) DMSO and inoculum were included in each experiment. The

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plates containing antibiotics were covered and incubated for 16 h at 37 ºC, while plates

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containing cinnamaldehye were covered and sealed with parafilm to prevent evaporation.

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These plates were incubated at 37 ºC for 16 to 18 h with shaking at 150 rpm (Titer Plate

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Shaker; Barnstead International, Dubuque, IA, USA) (Palaniappan & Holley, 2010).

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Then, wells were examined for turbidity and 40 µl of p-iodonitrotetrazolium violet (INT,

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Sigma-Aldrich) solution (0.2 mg/ml) was added to each well, incubated for 1 to 2 h at 37

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ºC to detect the presence of metabolic activity (Langfield, et al., 2004). The lowest

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concentration at which no red colour appeared was considered as the MIC. The

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experiment was conducted in triplicate and repeated twice for each antibiotic and

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cinnamaldehye. The resistance of E. coli to antibiotics (MIC’s > breakpoint values) was

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determined based on the reference values provided by CLSI (2002, 2007). Antibiotics to

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which E. coli strains were shown resistant were selected for studying interactive effects.

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2.4. Testing interaction between antibiotics and cinnamaldehyde

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The interactive inhibition by antibiotics and cinnamaldehyde was determined using the

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checkerboard method in combination with calculation of the fractional inhibitory

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concentration (FIC) index, which is commonly used for determination of synergy

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between antimicrobial compounds (Rand, Houck, Brown, & Bennett, 1993; White,

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Burgess, Manduru, & Bosso, 1996). Antibiotics (drug A) to which E. coli strains showed

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resistance were chosen for synergistic interaction testing with cinnamaldehyde (drug B).

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Each antibiotic-cinnamaldehyde combination was tested in triplicate and repeated twice.

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The antimicrobial agents were serially diluted in two-fold steps starting from their MIC

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values in 96 well microplates. A 50 µl aliquot of diluted inoculum was added and

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incubated for 16 h at 37 ºC. The inhibitory effects of combinations were examined by

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calculating the FIC index for each combination using the formula: FIC of drug A = MIC

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of drug A in combination/ MIC of drug A alone; FIC of drug B = MIC of drug B in

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combination/ MIC of drug B alone; FIC index = FIC of drug A + FIC of drug B (Pillai,

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Moellering, & Eliopoulos, 2005; Vigil, Palou, Parish, & Davidson, 2005). Interactive

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effects were defined as synergistic when the FIC index was ≤ 0.5. FIC indices > 0.5 and

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≤ 1 were considered additive; when between 1 and 2 they were considered without effect

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and when > 2 were considered antagonistic for the two antimicrobials (Langeveld, et al.,

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2014).

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3. Results and discussion

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E. coli ATCC 23739 and 02:0627 were resistant to tetracycline, while all strains were

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resistant to erythromycin and bacitracin (Table 1). All but E. coli ATCC 11775 were

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resistant to novobiocin. Although the MIC values of ampicillin and penicillin G for E.

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coli strains varied between 1 and 0.5 µg/ml, and 2 and 8 µg/ml, respectively, all strains

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were sensitive (< 32 µg/ml, CLSI break point) to these antibiotics (CLSI, 2002, 2007).

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The MIC values of cinnamaldehyde for E. coli ATCC11775, 8WT and 02:0627 were

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similar (Table 2), while for E. coli ATCC 23739 it was 800 µg/ml. The MIC values of

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cinnamaldehyde for E. coli 8WT and 02:0627 were consistent with previous findings

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(Visvalingam & Holley, 2012), where the MIC was determined using a broth

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macrodilution assay. These results suggest that like the MIC values of antibiotics, the

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MIC value of cinnamaldehyde for E. coli may vary between strains.

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Cinnamaldehyde enhanced the susceptibility of E. coli ATCC 23739 to tetracycline with

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an FIC value of 0.3 (Table 3). The susceptibility of all E. coli strains to erythromycin was

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synergistically enhanced by cinnamaldehyde (FIC ≤ 0.5), while it synergistically and

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additively enhanced the susceptibility of E. coli ATCC 23739 plus 02:0627 (FIC ≤ 0.5),

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and E. coli 8WT (FIC = 1.0) to novobiocin, respectively. Previously, Palaniappan and

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Holley (2010) reported that cinnamaldehyde increased the susceptibility of drug resistant

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E. coli N00 666 and S. Typhimurium SGI 1 (tet A) to erythromycin, tetracycline,

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novobiocin, ampicillin and penicillin G. Similarly, Johny, Hoagland, and

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Venkitanarayanan (2010) reported that cinnamaldehyde enhanced the susceptibility of

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drug resistant S. Typhimurium DT104 to ampicillin, streptomycin, sulfamethoxazole, and

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tetracycline. Cinnamaldehyde did not enhance susceptibility of tested E. coli strains to

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bacitracin (FIC index >1),which was consistent with the results of Palaniappan and

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Holley (2010). Findings of the current study and previous reports suggest that

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cinnamaldehyde could be used as an effective compound to enhance antibiotic

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susceptibility of E. coli. However, it is highly likely that successful inhibitory interaction

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between antibiotics and cinnamaldehyde against E. coli will depend on the nature and

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extent of antibiotic resistance originally present in the strain(s) of concern.

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Since the use of antibiotic growth promoters in animal production has been considered

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an important factor that has contributed to the emergence and spread of antibiotic

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resistant bacteria (Mathew, et al., 2007), several recent studies have examined the use of

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cinnamaldehyde or cinnamon oil, in which cinnamaldehyde is a major component, as an

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alternative to antibiotic-based growth promoters (Geraci, Garciarena, Gagliostro,

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Beauchemin, & Colombatto, 2012; Khorrami, Vakili, Mesgaran, & Klevenhusen, 2015;

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Vakili, Khorrami, Mesgaran, & Parand, 2013; Yan & Kim, 2012). Feeding growing pigs

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with a diet containing cinnamaldehyde or eugenol was found to have no effect on growth

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performance, but reduced the number of fecal E. coli and the noxious gas content of feces

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(Yan & Kim, 2012). Feeding beef cattle with a diet containing cinnamon oil or the

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antibiotic monensin was reported to have a similar effect on dry matter intake, apparent

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digestibility of nutrients and to reduce the ruminal population of protozoa and

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methanogens (Khorrami, et al., 2015). In another study, it was found that growth

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performance of feedlot cattle that were fed a diet containing cinnamaldehyde, eugenol

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and capsicum oleoresin was equivalent to cattle that were fed monensin (Geraci, et al.,

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2012).

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Overall, results from this study and feeding trails conducted with diets containing

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cinnamaldehyde suggest that cinnamaldehyde may be used as a potential antimicrobial

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agent to control antibiotic resistant E. coli in livestock production without affecting

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growth performance of animals. However, the observed variability in the susceptibility of

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E. coli strains to cinnamaldehyde as well as antibiotic-cinnamaldehyde combinations

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suggest that more studies need to be conducted to include a large number of E. coli

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strains with different environmental backgrounds. It is evident that as further regulatory

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restrictions on antibiotic use in agriculture occur, alternatives like cinnamaldehyde with

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potent inhibitory effects will become of greater importance in livestock production. Thus,

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further studies are also needed to evaluate the relationship between feeding natural

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antimicrobial agents and the prevalence of antibiotic resistant bacteria in livestock

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production.

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Acknowledgement

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Financial support for this study was provided by the Natural Sciences and Engineering

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Research Council of Canada.

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Table 1. The MIC values of antibiotics against four Escherichia coli strains. E. coli strain

MIC of Antibiotics (µg/ml)* PenicillinG Ampicillin Tetracycline Erythromycin Novobiocin Bacitracin

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ATCC 11775

4 (S)

0.5 (S)

0.5 (S)

16 (R)

32 (S)

>512( R)

ATCC 23739

2 (S)

0.5 (S)

32 (R)

32 (R)

128 (R)

>512 (R)

8WT

8 (S)

1.0 (S)

0.5 (S)

64 (R)

64 (R)

>512 (R)

02:0627

4 (S)

0.5 (S)

16 (R)

16 (R)

128 (R)

>512 (R)

*S – sensitive, R – resistant.

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Table 2. The MIC values of cinnamaldehyde against four Escherichia coli strains. E. coli strain ATCC 11775

MIC (µg/ml) 400

ATCC 23739

800

8WT

400

02:0627

400

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Table 3. Combined effect of antibiotics and cinnamaldehyde expressed as fractional inhibitory concentration (FIC) indexa FIC index E. coli strain ATCC ATCC 8WT 02:0627 11775 23739 Erythromycin 0.5 0.3 0.5 0.5 Tetracycline -b 0.3 Novobiocin 0.2 1.0 0.5 Bacitracin >1 >1 >1 >1 a Synergy = an FIC index ≤ 0.5; additive = an FIC index between > 0.5 ≤ 1.0; no effect = > 1 ≤ 2; antagonism = an FIC index > 2.0. b Not tested. Antibiotics

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