Bactericidal effects of Cinnamon cassia oil against bovine mastitis bacterial pathogens

Food Control 66 (2016) 291e299

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Bactericidal effects of Cinnamon cassia oil against bovine mastitis bacterial pathogens Hongmei Zhu a, c, Min Du b, Larry Fox b, Mei-Jun Zhu a, * a

School of Food Science, Washington State University, Pullman, WA 99164, USA Department of Animal Science, Washington State University, Pullman, WA 99164, USA c College of Food Science, Shanxi Normal University, Shanxi, 041004, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 November 2015 Received in revised form 9 February 2016 Accepted 11 February 2016 Available online 15 February 2016

Organic food production is expanding rapidly. However, this industry is hampered by the lack of effective antimicrobial agents which can be used in organic food production. This study examined the antimicrobial activity of Cinnamon cassia oil against major pathogens causing bacterial bovine mastitis, its miscibility in milk and possible antimicrobial mechanisms. C. cassia oil had inhibitory activity against all tested pathogen isolates from bovine mastitis. We conducted disk diffusion assay and found that discs with 20 mL of 2% (v/v) C. cassia oil solution resulted in inhibition zones of 29.6, 19.1, 27.0, 33.3 and 30.7 mm for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus xylosus and Escherichia coli 29, respectively. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of C. cassia oil was 0.00625% and 0.025% (v/v) for S. hyicus, 0.025% and 0.10% (v/v) for E. coli 29, and 0.0125% and 0.05% for S. aureus, S. epidermidis and S. xylosus, respectively. We selected two common mastitis pathogens, a representative S. aureus isolate and E. coli 29 for further analyses. Based on time-kill assay in LB broth with 0.15% agar, 2MBC of C. cassia oil generated bactericidal effects on S. aureus and E. coli 29 within 30 min, and 4MBC caused 6 log reduction of S. aureus and E. coli 29 within 30 min. In milk, C. cassia oil at 4MBC reduced ~6.0 Log10 CFU/ml of S. aureus and E. coli 29 to undetectable level within 8 h. Using propidium iodide staining, we observed membrane damage on both S. aureus and E. coli 29 cells during incubation with C. cassia oil. In addition, C. cassia oil treatment at MIC impaired membrane integrity of E. coli and S. aureus, which was followed by a decrease in ATP synthesis. Bacterial extracellular signaling quorum sensing orchestrates important events related to bacterial pathogeneses through excreting autoinducer (AI). Sub-inhibitory concentration of C. cassia oil repressed AI-2, a universal signal molecule mediating quorum sensing, production in S. aureus and E. coli 29 isolates. Collectively, our data show that C. cassia oil provides an exciting potential to be used as an alternative antimicrobial for bovine mastitis in organic dairy farms. Published by Elsevier Ltd.

Keywords: Cinnamomum cassia oil Bovine mastitis S. aureus E. coli Antimicrobial Organic farm

1. Introduction Mastitis is the most prevalent disease with huge economic impact on the dairy industry worldwide, due to loss related to discarded milk, lack of effective treatments, animal welfare concerns as well as potential public health consequence. A diverse group of pathogens have been implicated in bovine mastitis, which include bacteria, mycobacteria, mycoplasmas, yeast and fungi, and even algae, with bacteria to be the primary cause (Watts, 1988). Generally speaking, Staphylococci including Staphylococcus aureus

* Corresponding author. E-mail address: [email protected] (M.-J. Zhu). http://dx.doi.org/10.1016/j.foodcont.2016.02.013 0956-7135/Published by Elsevier Ltd.

and coagulase negative Staphylococcus such as Staphylococcus chromogenes, Staphylococcus epidermidis and Staphylococcus hyicus are the most common pathogens causing bovine mastitis followed by Streptococci and Escherichia coli (Contreras & Rodriguez, 2011). Given that mastitis can be caused by a diverse group of pathogens, control and cure of mastitis is difficult and challenging. In conventional dairy production, intra-mammary antibiotic infusion is the most common and conservative practice for treating clinical bovine mastitis. However, it usually has a variable efficacy, and has the potential to induce antibiotic resistance in bacterial strains (McDougall, Hussein, & Petrovski, 2014; Wichmann, UdikovicKolic, Andrew, & Handelsman, 2014), negatively impacting water

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and soil environments. Thus, there is a need for alternative nonantibiotic treatment strategies or approaches for controlling mastitis in dairy bovines. These strategies include developing new vaccines (Pereira, Oliveira, Mesquita, Costa, & Pereira, 2011), implementing good management systems and others (McDougall, Parker, Heuer, & Compton, 2009). Cinnamon cassia oil is a natural plant-derived essential oil that has been traditionally used to preserve foods as well as enhance food flavor. It is generally recognized as safe (GRAS), and does not cause resistance even after prolonged exposure (Becerril, Nerin, & Gomez-Lus, 2012). In addition, cinnamon oil demonstrates its inhibitory effects against a broad range of bacterial pathogens (Sheng & Zhu, 2014; Todd, Friedman, Patel, Jaroni, & Ravishankar, 2013). Up to now, however, the effectiveness of cinnamon oil in treating cow mastitis has not been tested. In this study, we tested the antimicrobial efficacy of C. cassia oil in inhibiting the growth of bovine mastitis isolates in the pure culture media as well as in milk. Milk has a complex food matrix where endogenous proteins, fat and other nutrients may protect mastitis pathogens or interfere with antimicrobial molecules, thereby demonstrating the antimicrobial activity of C. cassia oil in milk is essential for its use as an antimicrobial agent for intramammary infusion to treat mastitis. Quorum sensing (QS) mediates bacterial cell-to-cell communication via excreting signal molecules known as autoinducer (AI). Of which, AI-2 is a universal QS signal molecule, which regulates motility and Shiga toxin production in E. coli O157:H7 (Sperandio, Torres, Giron, & Kaper, 2001), capsular polysaccharide synthesis related gene expression in S. aureus (Zhao, Xue, Shang, Sun, & Sun, 2010) and antibiotic susceptibility in Streptococcus anginosus (Ahmed, Petersen, & Scheie, 2007). Thus, we further explored the mode of antimicrobial action of C. cassia oil as well as its impacts on the production of AI-2, focusing on S. aureus and E. coli. 2. Materials and methods 2.1. Cinnamon oil and bacterial strain C. cassia oil was purchased from Sigma/Aldrich (St. Louis, MO). The mastitis isolates were cultivated from cases of mastitis, subclinical and clinical. Strains used in this study were listed in Table 1.

20 mL DMSO (3%, v/v) with 0.15% (v/v) Tween 80. The plates were left under BSL2 cabinet for 30 min at room temperature and then incubated at 37
C for 24 h. The diameters of the inhibition zones were measured in millimeters. Measurements were performed in triplicates. Data are given as means ± SEM of three independent experiments. 2.3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) Two-fold microdilution broth method was used to determine MIC of C. cassia oil against mastitis bovine isolates in 96 well plate format (Sheng & Zhu, 2014). Briefly, Mueller-Hinton broth containing 0.15% (w/v) agar (MHBA) (Mann & Markham, 1998) was used with C. cassia oil at concentrations varying from 0.16% to 0.003125% (v/v). Overnight bacterial cultures were washed with 1  PBS and diluted to approximately 1  106 CFU/mL in MHBA. Each well was inoculated with 5  105 CFU/mL of respective bacterial culture. Wells contained 200 mL of uninoculated MHBA were used as blanks. Wells had 5  105 CFU/mL of respective bacterial culture in MHBA without C. cassia oil were used as positive control. The plates were incubated statically at 37
C for 24 h. MIC was determined as the lowest concentration of C. cassia oil without visible growth (no change in turbidity) of bacteria. Six replicates were performed for each concentration. MBC is defined as the lowest concentration that inhibits bacterial growth on the LB agar plates (<10 CFU/plate was regarded as no growth) (Sheng & Zhu, 2014). Experiments were performed in triplicates. 2.4. Kill curves of S. aureus or E. coli 29 in LB broth Different concentrations of C. cassia oil (0e0.4%, v/v) were prepared in sterilized LB broth containing 0.15% (w/v) agar (LBA) and incubated in 37
C water bath till use. Overnight S. aureus or E. coli 29 culture was washed once in 1 PBS and 1:100 inoculated to the corresponding C. cassia oil solutions, mixed and incubated at 37
C statically for 0, 15, 30, 60, and 120 min. The survival of S. aureus or E. coli 29 at a specific C. cassia oil concentration and incubation time was enumerated by serial dilution and plating onto LB agar. The experiment was conducted independently for three times.

2.2. Disc diffusion assay

2.5. Miscibility with milk and bactericidal kinetics against S. aureus and E. coli 29

Disc diffusion method was employed to assess antibacterial activity of C. cassia oil (Sheng & Zhu, 2014). Tested bacteria were activated in LB for 8 h at 37
C, then sub-cultured at 1:1000 in LB for another 14 h at 37
C. Cultures were adjusted to 1  105 CFU/mL with sterile phosphate buffered saline (PBS, pH7.4). 100 mL of the suspension was spread over the Mueller-Hinton agar plate. C. cassia oil was 1:50 diluted in 3% (v/v) DMSO with 0.15% (v/v) Tween 80 in PBS and filter sterilized. Under aseptic conditions, sterile paper discs (Whatman no.5, 7 mm dia) were placed on the agar surface, and 5, 10, and 20 mL of 2% (v/v) C. cassia oil was applied onto the corresponding paper discs. Control paper disc was loaded with

Pasteurized 2% milk samples were purchased from a local grocery store and mixed with C. cassia oil at 0%, 0.1%, 0.2% or 0.4% for S. aureus or 0%, 0.2%, 0.4% or 0.8% for E. coli 29, respectively. Overnight S. aureus or E. coli 29 culture was washed in 1PBS and 1:100 inoculated to the corresponding C. cassia oil milk solutions, mixed and incubated at 39
C statically for 32 h. The survival of S. aureus or E. coli 29 in a particular C. cassia oil milk solution sampled at 4, 8, 16 and 32 h of incubation was enumerated by serial dilution in PBS and plating onto LB agar. The experiment was repeated independently three times. 2.6. HPLC analysis of ATP

Table 1 Pathogenic bovine mastitis isolates used in this study.

S. aureus S. epidermidis S. hyicus S. xylosus E. coli 29

Isolate

Origin

Source

91.48 228.51 106.51 205.4 260.29

Bovine Bovine Bovine Bovine Bovine

(Roberson, Fox, Hancock, & Besser, 1992) (Quirk et al., 2012) (Roberson et al., 1992) (Park et al., 2011) (This study)

Activated S. aureus or E. coli 29 cultures was washed once with sterile 1 PBS buffer. The bacterial culture was subjected to ATP depletion in 1PBS without glucose, energization or C. cassia oil treatment by 1:20 dilution in 1PBS with 1.0% (w/v) glucose containing 0%, ½ MIC (0.025% for E. coli 29 and 0.0125% for S. aureus) and MIC (0.05% for E. coli 29 and 0.025% for S. aureus) of C. cassia oil, respectively at RT for 1 h. Then, formaldehyde was added to each

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bacterial culture to a final concentration of 0.6% (w/v). Treated bacterial suspensions were centrifuged at 10,000  g for 5 min. The resulting bacterial pellet was homogenized in 0.1 M KOH solution, sonicated for 3 min and incubated on ice for 30 min. After centrifugation, the supernatant was neutralized with H3PO4. Supernatant was filtered with a 0.45 mm PTFE filter and then analyzed quantitatively by Shimadzu HPLC series (LC-20AD, pumps with a diode-assay detector (SPD-M30A), SHIMADZU, Kyoto, JAPAN) using Luna C18 (2) column (250  4.6 mm i.d.; 5 mm particle size) (Phenomenex, Torrance, CA, USA). The mobile phase consisted of 60 mM K2HPO4 and 40 mM KH2PO4 (elute A) and 100% Methanol (elute B). The gradient elution was at the flow rate of 1 mL/min with the following program: the initial ratio of B was 0% and kept for 10 min; raised to 10% in 20 min, 20% in 25 min and held for 2 min, and then returned to 0% in 1 min and maintained for 7 min. Detection was performed by a SPD-M30A detector set at a wavelength of 260 nm at 25
C. Analyte contents were quantified based on peak areas of ATP (Sigma, St. Louis, MO) standard.

were inoculated and cultured in LB broth with 0.5% (w/v) glucose with 0, ½ MIC, ¾ MIC, or MIC concentrations of C. cassia oil at 37
C with aeration for 6 h. The bacterial cultures were centrifuged at 12,000  g for 5 min. Cell free supernatant was collected and incubated with V. harveyi reporter strain BB170, which was 1:3000 diluted of overnight culture in AB medium in 96-well microtiter plates at 30
C. Light production was measured hourly using a BioTek microplate reader (Synergy H1, BioTek). This experiment was repeated three times independently. In addition, CFU of bacterial cultures at 6 h post C. cassia oil incubation were serial diluted and enumerated onto LB agar plates.

2.7. Fluorescence-activated cell sorting (FACS)

3. Results

Flow cytometry evaluation of bacterial cells following C. cassia oil treatment was conducted per a published method (Bouhdid et al., 2010). Briefly, S. aureus or E. coli 29 cultures was twice activated in LB broth, washed with sterile 1 PBS and adjusted to 1  107 CFU/mL in PBS, then subjected to 0, ½ MIC (0.025% for E. coli 29 and 0.0125% for S. aureus), or MIC (0.05% for E. coli 29 and 0.025% for S. aureus) of C. cassia oil treatment for 1 h at RT, when bacterial cells were centrifuged at 8,000  g for 5 min, re-suspended in 50 ml of Propidium Iodide (PI) solution (eBioscience, Hatfield, AL) for 15 min. Stained cells were 1:10 diluted in PBS buffer then sorted by guava esayCyte™ Flow Cytometers (Millipore, Billerica, MA). PI binds to double stranded DNA and gives red fluorescent color, but it is impermeable to the cell membrane thus is excluded by cells with intact plasma membranes. Cells stained with PI had irreversibly damaged membrane structure, corresponding to dead cells. Bacteria incubated with PBS were used for a negative control.

3.1. Antibacterial activity of C. cassia oil against bovine mastitis isolates

2.8. Transmission electron microscopy Twice activated S. aureus or E. coli 29 cultures at 37
C were washed twice with sterile 1 PBS buffer, and diluted to 1  107 CFU/ mL. The S. aureus or E. coli 29 bacterial suspension were then subjected to a final concentration of 0.0125% (v/v) or 0.025% (v/v) C. cassia oil treatment at 37
C with aeration for 3 h, respectively. S. aureus or E. coli 29 suspension without treatment were used as a control. After C. cassia oil treatment, cells were centrifuged at 5,000  g for 5 min. The resulting cell pellets were fixed by 2.5% (w/ v) gluteraldehyde (Sigma) for 24 h at 4
C, followed by rinsed with 1  PBS buffer three times. It was further fixed in 1% (w/v) osmium tetroxide solution for 24 h at 4
C. The bacterial samples were then dehydrated in gradient ethanol series (30%, 50%, 70%, 95% and 100%), each 10 min, followed by 10 min of acetone dehydration, Spurr's resin (Electron Microscopy Sciences (EMS), Hatfield, PA) infiltration and embedding. Ultrathin sections (~70 nm) were cut using a Reichert ultra-microtome (Leica Microsystems Inc., Chicago, IL), loaded onto the copper grid, and stained with 1% aqueous uranyl acetate for 20 min and Reynolds lead for 6 min. The images were observed on a T20 transmission electron microscope (FEI, Hillsboro, Oregon), operating at 200 kV. 2.9. Autoinducer-2 (AI-2) assay AI-2 assay was performed as described previously (Surette & Bassler, 1998). Briefly, ~1  107 CFU/mL S. aureus or E. coli 29

2.10. Statistical analysis Data were analyzed as a complete randomized design using GLM (General Linear Model of Statistical Analysis System, SAS, 2000). Mean ± standard errors of mean (SEM) are reported. Statistical significance is considered as P < 0.05.

The antibacterial inhibition zone of C. cassia oil to bacterial pathogen of bovine mastitis was assayed by disc diffusion assay. C. cassia oil had an antimicrobial effect against all tested bovine mastitis isolated with more prominent effects against S. aureus, S. xylosus and E. coli 29. For all tested strains, the inhibitory effect strengthened progressively with increasing amount of C. cassia oil per disc (Table 2). Discs with 20 mL of 2% (v/v) C. cassia oil solution resulted in inhibition zones of 29.6, 19.1, 27.0, 33.3 and 30.7 mm for S. aureus, S. epidermidis, S. hyicus, S. xylosus and E. coli 29, respectively. Vehicle control (3%, v/v DMSO with 0.15%, v/v Tween 80) had no inhibitory effect on all tested isolates (Table 2). The MIC and MBC of C. cassia oil was 0.00625% and 0.025% (v/v) for S. hyicus, 0.025% and 0.10% (v/v) for E. coli 29, and 0.0125% and 0.05% for S. aureus, S. epidermidis and S. xylosus, respectively (Table 3). C. cassia oil appeared to be more effective against S. aureus than against E. coli 29 (Table 3). 3.2. The bactericidal kinetics against S. aureus and E. coli 29 in culture media and milk The survival curve (Fig. 1A and B) showed that a 0.1% and 0.2% solution of C. cassia oil in LB broth, respectively, had bactericidal effects on S. aureus and E. coli 29 within 30 min. C. cassia oil at 4 MBC (0.2% and 0.4% for S. aureus and E. coli 29, respectively) caused 6 log reduction of S. aureus and E. coli 29 within 30 min (Fig. 1A and B). We further tested the miscibility and antimicrobial activity of C. cassia oil in milk, which provides essential information for its application in bovine mastitis. 0.2% and 0.4% solution of C. cassia caused a ~6.0 Log10 CFU/mL of S. aureus and E. coli 29, respectively, Table 2 Inhibitory zone of C. cassia oil against cow mastitis isolates (mm). Volume of 2% (v/v) C. cassia oil per disc (mL)

S. aureus S. epidermidis S. hyicus S. xylosus E. coli 29

0

5

ND ND ND ND ND

15.0 7.2 7.2 10.1 14.2

10 ± ± ± ± ±

0.3 0.0 0.0 0.6 0.3

20.0 9.1 12.5 17.5 21.7

Mean ± SEM of disc diffusion, n ¼ 3. ND: not detected.

20 ± ± ± ± ±

0.6 0.0 0.1 0.8 0.4

29.6 19.1 27.0 33.3 30.7

± ± ± ± ±

0.1 0.0 0.0 1.8 0.8

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Table 3 MIC and MBC of C. cassia oil against bovine mastitis isolates.

S. aureus S. epidermidis S. hyicus S. xylosus E. coli 29 MIC: Minimum Concentration.

Inhibitory

MIC (%, v/v)

MBC (%, v/v)

0.0125 0.0125 0.00625 0.0125 0.025

0.05 0.05 0.025 0.05 0.10

Concentration;

MBC:

Minimum

Bactericidal

control cells without C. cassia oil treatment had 8.7% of PI-stained cells (Fig. 3B). After 60 min incubation in PBS with C. cassia oil at the MIC and MBC level, the PI-stained cells were increased to 34.5% and 95.5%, respectively (Fig. 3B), clearly showing that C. cassia oil led to loss of membrane impermeability in both S. aureus and E. coli 29. The membrane changes were greater in E. coli 29 than that in S. aureus when both strains were exposed to their respective MIC and MBC values. TEM observation was further performed for S. aureus and E. coli 29 that have been treated with C. cassia oil for

Fig. 1. Survival curves of bovine mastitis isolates in LBA broth or milk containing different concentrations of C. cassia oil. A. E. coli 29 in LBA broth; B. S. aureus in LBA broth; C. E. coli 29 in milk; D. S. aureus in milk. Mean ± SEM, n ¼ 3.

to be reduced to an undetectable level within 8 h (Fig. 1C and D). 3.3. Inhibitory effects on ATP generation

3 h. Consistently, deformities with ruptured membrane was observed in both E. coli and S. aureus cells treated with C. cassia oil at the MIC level (Fig. 4).

Effects of C. cassia oil on ATP levels were next determined for S. aureus and E. coli 29. When 1.0% glucose was supplemented to S. aureus and E. coli 29 cells in PBS buffer (pH7.0) following 1 h incubation at room temperature, the cellular ATP was boosted dramatically in both S. aureus and E. coli 29 compared with cells without glucose addition (Fig. 2). However, treatment of energized S. aureus and E. coli 29 with ½ MIC or MIC level of C. cassia oil impaired or even prevented the cellular ATP increase in both S. aureus and E. coli 29 (Fig. 2B and C), with a more prominent effect on E. coli 29 cells (Fig. 2C).

3.5. Sub-inhibitory C. cassia oil suppresses universal autoinducer-2 production

3.4. Impacts on membrane integrity and cell morphology

Mastitis in organic dairy cattle can be a serious issue due to its prevalence and the lack of effective treatments. There is a demand for organic antimicrobials, which can be used for organic food production that do not induce development of antibiotic resistance and it appears that the risk of antibiotic resistant mastitis pathogens is reduced as a dairy herd transitions from conventional to organic status (Park, Fox, Hancock, McMahan, & Park, 2012). C. cassia oil had effective bactericidal activity against the pathogen

The alteration of membrane integrity was further assessed using membrane impermeable nucleic acid stain PI. In S. aureus without C. cassia oil treatment, almost all cells (only 0.4% of PI-stained cells) were impermeable to PI (Fig. 3A). After 60 min treatment with C. cassia oil at the MIC and MBC level, the PI-stained cells were augmented to 28.95% and 71.6%, respectively (Fig. 3A). In E. coli 29,

Besides its bactericidal effects at higher concentration, subinhibitory C. cassia oil at ½ 1 MIC inhibited AI-2 production in both E. coli and S. aureus cells (Fig. 5). C. cassia oil at ½ MIC had no inhibitory effects against the growth of S. aureus and E. coli, though ¾ MIC and 1 MIC inhibited their growth (Fig. 5A and B). 4. Discussion

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Fig. 2. Intracellular ATP concentration of bovine mastitis isolates in response to C. cassia oil treatment. A. A representative HPLC chromatographic profiles; B. S. aureus; C. E. coli 29. Mean ± SEM, n ¼ 3. Different letters represented a significant difference at P < 0.05. Peak area: area under ATP peak. Depletion: Bacteria were subjected to ATP depletion; Energy: Bacteria were subjected to 1.0% (v/v) glucose; Energy þ ½ MIC (E þ ½ MIC): Bacteria were subjected to 1.0% (v/v) glucose plus ½ MIC of C. cassia oil; Energy þ MIC (E þ MIC): Bacteria were subjected to 1.0% (v/v) glucose plus MIC of C. cassia oil.

types tested in this study, E. coli, S. aureus, S. epdermidis, S. hyicus and S. xylosus, which were the common pathogen types on organic dairies studied (Park et al., 2012). This was not unexpected as cinnamon oil has been reported to has antibacterial activity against bacterial pathogens (Unlu, Ergene, Unlu, Zeytinoglu, & Vural, 2010) including Listeria monocytogenes (Gill & Holley, 2004), non-O157 STEC (Sheng & Zhu, 2014), Pseudomonas sp. and S. aureus (Bouhdid et al., 2010; Kavanaugh & Ribbeck, 2012; Unlu et al., 2010), and S. epidermidis (Nuryastuti et al., 2009), which prompted us to hypothesize that C. cassia oil is an effective antimicrobial agent against pathogens causing bovine mastitis.

4.1. Antibacterial activity of C. cassia oil against bovine mastitis isolates In this study, we found that C. cassia oil was effective in killing all tested bacterial pathogens isolated from mastitic bovines. Overall, MIC as well as MBC within Staphylococcus sp. were very similar. MIC of C. cassia oil for S. aureus observed in this study was much lower than that of Cinnamomum verum essential oil (0.125%, v/v) (Bouhdid et al., 2010) or Cinnamomum zeylanicum essential oil (0.056%, v/v) (Unlu et al., 2010). The difference might be due to the variability of Cinnamon essential oil composition and/or strain susceptibility. We also found that E. coli isolate had a higher MIC and MBC than those of Staphylococcus sp. indicating that C. cassia oil is more effective against Gram þ Staphylococci than Gram - E. coli. The difference in effectiveness is probably due to the difference in membrane structure and composition. Gram - bacteria possess an outer

membrane that serves as an impermeable barrier, thus preventing C. cassia oil from penetrating into cells. In addition, extracellular enzymes existing in the periplasmic space might hydrolyze antimicrobial component in C. cassia oil, further reducing its antimicrobial activities. In supporting of our finding, the previous study reported that oregano essential oil had a higher MIC against Gram e Pseudomonas aeruginosa than S. aureus (Bouhdid, Abrini, Zhiri, Espuny, & Manresa, 2009). Similar to our finding, MIC of C. zeylanicum Blume against E. coli is one fold higher than that of S. aureus (Unlu et al., 2010). However, trans-cinnamaldehyde, the major antimicrobial of C. cassia oil, showed the same MIC and MBC against S. aureus and E. coli in milk and a similar Time-Kill activity (Baskaran, Kazmer, Hinckley, Andrew, & Venkitanarayanan, 2009). Interestingly, we found that C. cassia oil was more effective against E. coli than S. aureus at their respective MIC in pure media setting. For its application in treating infection in mastitis bovine, C. cassia oil needs to be miscible with milk and achieves antimicrobial activity in complex matrix in vivo. Our data indicated that C. cassia oil at 4 MBC can effectively eliminate both S. aureus and E. coli 29 in milk within 8 h. C. cassia oil had similar efficacy to E. coli and S. aureus in milk at their respective 4MBC, but was more effective than trans-cinnamaldehyde reported previously (Baskaran et al., 2009). Of note, cinnamon oil used at this concentration might negatively impact the flavor of milk. However, given the fact that cinnamon oil is used only to cure the mastitis, milk will not be used for human consumption during this period.

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Fig. 3. Effects of C. cassia oil on the membrane permeability of bovine mastitis isolates as assayed by fluorescence-activated cell sorting (FACS). A. S. aureus; B. E. coli 29. The relative propidium iodide intensities on the left of red line was taken as live cells, and those on the right of red line were considered as dead cells. CON: cells were incubated in PBS at RT for 1 h; MIC: Cell were incubated in MIC concentration of cinnamon oil (0.0125% for S. aureus and 0.025% for E. coli 29) at RT for 1 h; MBC: Cell were incubated in MBC concentration of cinnamon oil (0.05% for S. aureus and 0.1% for E. coli 29) at RT for 1 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 4. Transmission electron microscopic images of untreated and C. cassia oil treated bovine mastitis isolates, S. aureus and E. coli 29. A. untreated S. aureus control; B and C. S. aureus treated with C. cassia oil; D. untreated E. coli 29 control; E and F. E. coli 29 treated with C. cassia oil. Time of contact was 3 h and C. cassia oil concentration was MIC.

4.2. Action mode of C. cassia oil antimicrobial mechanism The main component of C. cassia oil used in this study was cinnamaldehyde (59.96%) (Sheng & Zhu, 2014). The antimicrobial effectiveness of C. cassia oil is comparable to cinnamaldehyde (Ooi et al., 2006). Cinnamaldehyde has hydrophobic property, thus having the ability to react with bacterial cell membranes, causing membrane damage. Indeed, based on flow cytometry examination, a short duration of C. cassia oil incubation at MIC or MBC levels led to enhanced PI penetration to both S. aureus and E. coli cells, implicating the oil's ability to cause lesions in cell membranes. However, the previous study reported that 1 h exposure to C. verum essential oil at the MIC level had minimal effect on PI uptake in S. aureus, though 1.5 MIC of C. verum essential oil treatment enhanced PI cell permeability in S. aureus (Bouhdid et al., 2010). Furthermore, we found that C. cassia oil treatment resulted in more membrane damage in E. coli than in S. aureus when they were exposed to their respective MIC and MBC levels of C. cassia oil, despite a higher MIC for E. coli compared to that for S. aureus. In line, oregano essential oil, had a higher MIC for P. aeruginosa compared to that for S. aureus, but caused more functional and ultrastructural changes in P. aeruginosa than in S. aureus when treated at their respective MICs (Bouhdid et al., 2009). Our finding that C. cassia oil impaired cytoplasmic membrane integrity was further supported by TEM observation. C. cassia oil at their respective MICs resulted in rapture of the cellular membrane or cell shape deformation, which was associated with decreased ATP synthesis, possibly due to disruption of membrane potential, given the importance of plasma membrane integrity in ATP generation in bacteria (Mora-Pale et al., 2015). Similar morphological

changes and cell deformation was observed when Enterococcus are treated with a citrus oil blend (Fisher & Phillips, 2009), and Oregano essential oil to P. aeruginosa and S. aureus (Bouhdid et al., 2009) and L. monocytogenes, Salmonella Typhimurium, and E. coli O157:H7 (Bhargava, Conti, da Rocha, & Zhang, 2015). Similarly, cinnamaldehyde treatment caused a rapid decline in cellular ATP levels in energized L. monocytogenes (Gill & Holley, 2004). Spanish oregano as well as C. cassia oil treatment at their MIC impairs the membrane integrity of E. coli O157:H7 and L. monocytogenes and induces intracellular ATP depletion (Oussalah, Caillet, & Lacroix, 2006), suggesting a toxic disruptive action on cytoplasmic membrane. This membrane structural damage is likely caused by the high affinity of essential oil to membrane lipid bilayers, which perturbs membrane structure (Seow, Yeo, Chung, & Yuk, 2014). In addition, essential oil can coagulate bacterial surface molecules, which also damages cell walls and membrane structure (Bouhdid et al., 2010). 4.3. Suppressing universal AI-2 production as an alternative strategy Mastitis in bovine can be caused by a diverse group of pathogenic bacteria, which makes it difficult and challenging in controlling and curing this disease. Finding a universal antimicrobial that has broad spectrum is important. Currently, intra-mammary antibiotic infusion is the most common practice for treating bovine mastitis in the conventional dairy production, which has the potential to induce antibiotic resistance in bacterial strains (McDougall et al., 2014; Wichmann et al., 2014), and demonstrates variable efficacy. AI-2 is a universal signal molecule mediating

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Fig. 5. Impacts of sub-inhibitory concentration of cinnamon oil on growth and autoinducer-2 production of bovine mastitis isolates, S. aureus and E. coli 29 during 6 h treatment at 37
C. A. CFU enumeration of S. aureus; B. CFU enumeration of E. coli 29; C. Autoinducer-2 production of S. aureus; D. Autoinducer-2 production of E. coli 29. Mean ± SEM; n ¼ 3.

bacterial QS, which is a global regulator of virulence of pathogenic bacteria (Ahmed et al., 2007; Sperandio et al., 2001; Zhao et al., 2010). However, only few studies tested effectiveness of cinnamon oil on AI-2 based QS (Brackman et al., 2008). C. cassia oil at sub-inhibitory concentration dramatically inhibits AI-2 production in S. aureus and E. coli without inhibiting their growth, providing another layer of protection to bovine mastitis. In supporting of our finding, Brackman et al. (2008) reported that cinnamaldehyde interferes with AI-2 based QS in various Vibrio spp., resulting in increased antibiotic susceptibility and reduced virulence (Brackman et al., 2008). Furthermore, cinnamon oil does not induce antimicrobial resistance of pathogens to either cinnamon itself or other antibiotics (Becerril et al., 2012), making C. cassia oil an alternative antimicrobial for treating bovine mastitis. In conclusion, C. cassia oil not only has bactericidal effects against bovine mastitis isolates when it was applied at higher concentration, but also is able to suppress AI-2 production used at sub-inhibitory concentration. We further found that C. cassia oil disrupts membrane structure to generate bactericidal effect. Thus, it has a great potential to be used as an alternative antibacterial agent to control bovine mastitis in organic farms. Competing financial interest The authors declare no competing financial interest. Acknowledgments We thank Dr. Shuming Zhang, Dr. Meng Zhao, Lina Sheng and Hsieh-Chin Tsai for their assistance in analyses. This activity was funded by an BioAg Research Competitive Grant from the Agricultural Research Center at Washington State University, College of Agricultural, Human, and Natural Resource Sciences.

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