Inhibition and Inactivation of <I>Listeria Monocytogenes</I> and <I>Escherichia Coli</I> O157:H7 Colony

2947

Journal of Food Protection, Vol. 69, No. 12, 2006, Pages 2947–2954 Copyright ᮊ, International Association for Food Protection

Inhibition and Inactivation of Listeria monocytogenes and Escherichia coli O157:H7 Colony Bioﬁlms by Micellar-Encapsulated Eugenol and Carvacrol

D. PE´ REZ-CONESA,1,2 L. MCLANDSBOROUGH,1 AND J. WEISS1*

1Department of Food Science, University of Massachusetts, 100 Holdsworth Way, Amherst, Massachusetts 01003, USA; and 2Nutricio´n y Bromatologı´a, Facultad de Veterinaria, Universidad de Murcia, Campus de Espinardo, 30071 Murcia, Spain Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/12/2947/2002115/0362-028x-69_12_2947.pdf by guest on 27 September 2021 MS 06-258: Received 9 May 2006/Accepted 8 July 2006

ABSTRACT

The antimicrobial efficacy of carvacrol and eugenol, two essential oil compounds, encapsulated in a micellar nonionic surfactant solution on four strains of Listeria monocytogenes (Scott A, 101, 108, and 310) and four strains of Escherichia coli O157:H7 (H1730, E0019, F4546, and 932) growing as colony biofilms was investigated. Carvacrol and eugenol were encapsulated in Surfynol 485W at concentrations ranging from 0.3 to 0.9% (wt/wt) at a surfactant concentration of 5% (wt/wt). Colony biofilms were grown on polycarbonate membranes resting on agar plates containing antimicrobial formulations. Cells were enumerated after 0, 3, 6, 9, 24, 48, and 72 h of incubation. Colony biofilms of all E. coli O157:H7 strains were more sensitive to both antimicrobial systems than L. monocytogenes strains. Surface-grown E. coli O157:H7 viable cell numbers decreased below detectable levels after exposure to encapsulated essential oil compounds for Ͼ3 h at all tested concentrations, except for E. coli O157:H7 F4546, which grew slowly in the presence of Ͻ0.5% (wt/wt) eugenol. L. monocytogenes Scott A and 101 were more resistant to eugenol than carvacrol at sublethal concentrations (Ͻ0.5% [wt/wt]). Carvacrol was effective at any concentration against L. monocytogenes 108, whereas concentrations of Ͼ0.5% (wt/wt) eugenol were required for inactivation. L. monocytogenes 310 was equally sensitive to both essential oil compounds. Results suggest that surfactant- encapsulated generally recognized as safe essential oil compounds may offer a new means to control the growth of food pathogens such as E. coli O157:H7 and L. monocytogenes on food contact surfaces.

Biofilms have been defined as matrix-enclosed micro- Microbial cells within biofilms and adhering single organisms that adhere to a surface and/or to each other, cells may exhibit higher resistance to antimicrobials and producing a dynamic environment in which the component sanitizers than their planktonic counterparts (2, 6, 19, 32, microbial cells appear to reach homeostasis, optimally or- 34, 37, 49). This resistance has been attributed to multiple ganized to make use of all available nutrients (32). Cells in factors, one of which is the presence of an extracellular the latter stages of the growth cycle may be embedded in polymeric substance matrix that encloses the microbial a matrix of extracellular polymeric substances and have a cells. Extracellular polymeric substances may act as a bar- different rate of growth, cellular morphology, and physi- rier against the penetration of sanitizers and antimicrobials, ology than their planktonic counterparts (13). Listeria thereby shielding embedded microbial cells (8). Other fac- monocytogenes and Escherichia coli O157:H7 are two im- tors include production of enzymes that might degrade an- portant foodborne pathogens that are able to form biofilms timicrobial compounds and an altered physiological state of on various surfaces (16, 17). In addition to growing on the cells in biofilms that leads to increased resistance to mem- surface of various foods, L. monocytogenes has been re- brane-perturbating or oxidizing compounds and tolerance ported to attach to stainless steel, plastic, polyester, Teflon, to variations in nutrient concentrations (4, 11, 12, 26, 30, and rubber surfaces (12, 43, 57), whereas E. coli O157:H7 43). can reportedly form biofilms on stainless steel (11, 38, 40). The antimicrobial activity of spices and herb extracts E. coli Insufficient control of surface growth of O157:H7 has been attributed to the presence of phenolic compounds and L. monocytogenes on foods and processing equipment in essential oils. Many authors have reported on the efficacy may lead to cross-contamination and postprocessing con- of phytophenols as antimicrobial agents, especially carva- tamination, leading to food spoilage and serious illnesses crol from oregano and thyme and eugenol from clove, re- (30). Listeriosis can be a life-threatening illness in immu- spectively (5, 20–23, 27–29). Phytophenols may disrupt the nocompromised and pregnant individuals. E. coli O157:H7 integrity of the cytoplasmatic membrane, thereby severely has been associated with hemorrhagic colitis, and may in some instances induce hemolytic uremic syndrome and limiting the cells’ ability to produce ATP and maintain ho- thrombotic thrombocytopenia purpura (36). meostasis (9, 42). Because of the hydrophobicity and low water solubility of phytophenols, their potential as antimi- * Author for correspondence. Tel: 413-545-1025; Fax: 413-545-1262; crobial agents against biofilms has rarely been exploited E-mail: [email protected]. (14). We previously demonstrated that encapsulation of 2948 PE´ REZ-CONESA ET AL. J. Food Prot., Vol. 69, No. 12 phytophenols in surfactant micelles can help overcome Technology culture collection and stored as stock cultures at these application challenges and lead to an effective control Ϫ70ЊC. Working cultures were maintained on slants and were in- system for growth of foodborne pathogens in planktonic oculated from frozen stocks monthly and stored at 4ЊC. For each cultures (21–23). It should be noted that eugenol and car- experiment, a loopful of the culture was transferred to tryptic soy vacrol cannot be dispersed at levels ranging from 0.3 to broth (TSB) or TSB/0.6% yeast extract (TSBYE) (Difco, Detroit, Mich.) for E. coli O157:H7 and L. monocytogenes, respectively, 0.9% (wt/wt) in aqueous phase systems in the absence of and incubated at 32ЊC for 24 h. Bacteria were then subcultured in a nonionic surfactant. Instead, polyphenolic droplets rapidly either TSB or TSBYE at 32ЊC for 18 h prior to exposure assays. increase in size due to Ostwald ripening and phase separate. Thus, testing of the antimicrobial in the absence of surfac- Preparation of agar media with antimicrobial-containing tant at these concentrations was not feasible (22, 23). micellar solutions. Micellar antimicrobial containing plates with The objective of this study was to assess the antimi- TSB for E. coli O157:H7 and TSBYE for L. monocytogenes were crobial activity of carvacrol and eugenol encapsulated in prepared as follows. One milliliter of either surfactant solution Surfynol 485W micelles against membrane-supported E. without antimicrobials or micellar eugenol or carvacrol solutions Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/12/2947/2002115/0362-028x-69_12_2947.pdf by guest on 27 September 2021 was aseptically mixed with 14 ml of autoclaved agar to give a coli O157:H7 and L. monocytogenes surface growth. Bac- final concentration of 5% for Surfynol 485W; 0.3, 0.5, and 0.9% terial colonies were grown on black polycarbonate filters for eugenol; and 0.3, 0.5, and 0.7% for carvacrol. Controls con- placed on agar plates. Colony biofilms have previously sisted of agar plates (TSB and TSBYE) without any additives or been used to study effectiveness and transport of antibiotics with 5% Surfynol 485W alone. For both essential oils, the anti- and antimicrobials into biofilms (2, 55, 56). In contrast to microbial concentrations used were below their respective maxi- biofilms cultured in a reactor (e.g., Centers for Disease mum additive concentrations, that is, the highest concentration of Control biofilm, rotating disk, or rotating annular reactor) antimicrobial that can be incorporated into a micellar surfactant in which bacteria are grown at a solid/liquid interface with solution at a given surfactant concentration (0.96% for eugenol diffusion of nutrients and antimicrobials from the aqueous and 0.7% for carvacrol at 5% Surfynol 485W) to ensure that no phase into the biofilm, colony biofilms are grown on a per- free antimicrobial was left in the aqueous phase (22, 23). meable substrate surface at the solid/air rather than at the Colony biofilm preparation. Colony biofilms were grown solid/liquid interface (32). Water, nutrients, and antimicro- on polycarbonate membrane filters resting on agar plates (2, 56). bials diffuse from the supporting agar through the perme- Black, polycarbonate membrane filters (diameter, 25 mm; pore able substrate surface into the bacterial biofilm. This pro- size, 0.2 ␮m; Poretics, Livermore, Calif.) were sterilized under cess is somewhat representative of bacterial growth on solid UV light for 15 min on each side and aseptically placed on TSB- food surfaces and was therefore selected to model surface or TSBYE-based media formulations for E. coli O157:H7 or Lis- growth and destruction in the presence of antimicrobial mi- teria monocytogenes, respectively. Overnight planktonic cultures celles. Moreover, the systems used in this study can be for- of L. monocytogenes or E. coli O157:H7 were diluted to an optical mulated with all generally recognized as safe compounds. density of 0.05 at 600 nm in TSBYE for L. monocytogenes or Although we chose to use a non–food-approved surfactant, TSB for E. coli O157:H7. The optical density was measured in a 1-cm path length quartz cuvette using a UV-visible spectropho- we have been able to manufacture these systems with food- tometer (Ultrospec 2000, Pharmacia Biotech, Cambridge, UK). approved surfactants as well (data not shown). One 5-␮l drop of diluted planktonic culture was used to inoculate each polycarbonate membrane resting on the agar culture medium MATERIAL AND METHODS giving an initial microbial load of approximately 105 CFU per Preparation of antimicrobial-containing Surfynol 485W membrane. Initial studies indicated that inoculation at this level micelles. Surfactant micelles containing eugenol and carvacrol allowed for monitoring of both increasing and decreasing cell were prepared by dispersing the nonionic surfactant Surfynol numbers in the presence of different concentrations of antimicro- 485W (Air Products and Chemical, Allentown, Pa.) in double- bial micelles and surfactant. The plates were inverted and incu- distilled and deionized water at room temperature to obtain a stock bated at 32ЊC for a total of 72 h. solution with a surfactant concentration of 75% (wt/wt) (22, 23). Eugenol (4-allyl-2-methoxyphenol) and carvacrol (5-isopropyl-2- Bacterial enumeration. Colony biofilms were sampled at 0, methyl-phenol), purchased from Sigma (St. Louis, Mo.), were 3, 6, 9, 24, 48, and 72 h. Two membrane-containing colonies were added under constant stirring to the surfactant solution to create removed from the agar culture medium with sterile forceps and stock solutions containing 4.5, 7.5, and 13.5%, and 4.5, 7.5, and placed in 10 ml of buffered peptone water (BPW; Difco). The 10.5% (wt/wt) of eugenol or carvacrol, respectively. To ensure tube was vortexed briskly for 2 min to remove cells from the complete encapsulation of antimicrobials in surfactant micelles, membrane, and the bacterial suspension was then serially diluted samples were withdrawn in regular intervals, and their UV-visible in BPW. Serially diluted samples were plated onto TSBYE agar absorption spectra were measured. As previously reported, solu- for L. monocytogenes and R2A agar (Becton Dickinson, Sparks, tions became transparent and adsorption at 300 to 800 nm de- Md.) for E. coli O157:H7 using an automated spiral plater (Spiral Њ creased to zero, indicating completion of the encapsulation pro- Biotech, Norwood, Mass.). The plates were incubated at 32 C for cess (22, 23). Solutions were filter sterilized using a 0.22-␮m cel- 24 h. R2A medium is a low nutrient medium used in water mi- lulose acetate membrane (Corning, Corning, N.Y.) and stored at crobiology for the recovery of stressed and chlorine-tolerant bac- 4to6ЊC until used. teria (15). It was used in this project because the colony size of E. coli O157:H7 was smaller on R2A than on high nutrient media Bacterial strains and growth conditions. Four strains each and allowed for more accurate counts of this organism using the of L. monocytogenes (Scott A, 101, 108, and 310) and E. coli spiral plater and automated counter. Larger sized colonies are O157:H7 (H1730, F4546, 932, and E0019) were obtained from more difficult to be accurately counted by the automated counter the University of Tennessee, Department of Food Science and since they tend to merge. After incubation at 32ЊC, the plates were J. Food Prot., Vol. 69, No. 12 INHIBITION OF L. MONOCYTOGENES AND E. COLI O157:H7 COLONY BIOFILMS 2949

FIGURE 1. Levels (log N/N0)ofL. monocytogenes (A) Scott A, (B) 101, (C) 108, and (D) 310 in the presence of 5% Surfyn- ol 485W and 0, 0.3, 0.5, and 0.9% eugenol as a function of incubation time at 0, 3, 6, 9, 24, 48, and 72 h. Control levels represent growth on tryptic soy agar with 0.6% yeast extract, and N0 was the level (CFU per membrane) of the control at T ϭ 0. The center dotted line indicates the initial level of bacterial cells (N0). The lower line indicates the minimum detectable level, and any points on this line indicate that

levels were below the detectable limit. ⅷ, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/12/2947/2002115/0362-028x-69_12_2947.pdf by guest on 27 September 2021 Control; ⅜, Surfynol; Ⅵ, eugenol (0.3%); ᭡, eugenol (0.5%); ᭞, eugenol (0.9%).

read using an automated counter (Q-Count, Spiral Biotech). Three against strain 310. L. monocytogenes strain 101 was least replicates of membranes were used and cell numbers were plotted susceptible to eugenol (Fig. 1B), showing growth similar as means with standard deviations. The minimum detectable level to that of the surfactant and controls in the presence of 0.3 by the Q-count automated counter was 40 CFU per membrane or and 0.5% eugenol. Cell numbers decreased by approxi- 2 8.16 CFU/cm . mately 2 log only upon prolonged exposure (Ͼ72 h) to RESULTS 0.9% eugenol. L. monocytogenes strain Scott A (Fig. 1A) also grew in the presence of 0.3 and 0.5% eugenol, but Surface growth of L. monocytogenes and E. coli in when treated with 0.9% eugenol, viable cells were no lon- the absence of antimicrobials. Colony biofilms for all ger detected after 48 h. L. monocytogenes strain 108 was strains of both microorganisms grew similarly well in the susceptible to 0.5 and 0.9% eugenol with a Ͼ4-log reduc- absence of antimicrobials and surfactant (controls) and in tion observed within the first 24 h of the treatment. How- the presence of 5% (wt/wt) of nonionic surfactant micelles ever, exposure to 0.3% eugenol led to bacteriostasis for the (Figs. 1 through 4). After inoculating the membranes, cell first 48 h, after which cell numbers increased by 2.2 log. numbers increased from approximately 105 to 109 CFU per Surface growth of four L. monocytogenes strains in membrane. Thus, empty Surfynol 485W micelles did not the presence of encapsulated carvacrol. The sensitivity have any inhibitory efficacy against colony biofilms when of L. monocytogenes to carvacrol-loaded micelles (0.3, 0.5, compared to the control. 0.7%) is shown in Figure 2. Carvacrol inhibited the growth Surface growth of four L. monocytogenes strains in of all four strains at all concentrations tested and reduced the presence of micellar-encapsulated eugenol. The sen- populations below detectable levels upon extended incu- sitivity of L. monocytogenes to eugenol-loaded micelles bation (72 h). Strain 310 was the most sensitive. After as (0.3, 0.5, 0.9%) varied significantly between strains (Fig. little as 3 h of exposure to 0.3% carvacrol, cell numbers 1). Among the L. monocytogenes strains tested, strain 310 decreased by 3.4 log, and were below detectable levels after was the most sensitive to eugenol (Fig. 1D). Cell numbers 24 h of treatment regardless of the antimicrobial concen- decreased by 3.7 log after 3 h of exposure to 0.5 and 0.9% tration used. Populations of strains Scott A, 101, and 108 eugenol. Strain 310 was no longer detectable after 9 h of decreased more slowly at all tested carvacrol concentra- exposure to micellar-encapsulated 0.3% eugenol. Eugenol tions, with these strains showing a 2.4- to 3.6-log reduction led to bacterial inactivation when used at 0.9% against within 48 h of treatment. strains Scott A (Fig. 1A) and 101 (Fig. 1B), at 0.5 and 0.9% Surface growth of four E. coli O157:H7 strains in against strain 108 (Fig. 1C), and at all tested concentrations the presence of encapsulated eugenol. Colony biofilm 2950 PE´ REZ-CONESA ET AL. J. Food Prot., Vol. 69, No. 12

FIGURE 2. Levels (log N/N0)ofL. monocytogenes (A) Scott A, (B) 101, (C) 108, and (D) 310 in the presence of 5% Surfy- nol 485W and 0, 0.3, 0.5, and 0.7% carvacrol as a function of incubation time at 0, 3, 6, 9, 24, 48, and 72 h. Control levels represent growth on tryptic soy agar with 0.6% yeast extract, and N0 was the level (CFU per membrane) of the control at T ϭ 0. The center dotted line indicates the initial level of bacterial cells (N0). The lower line indicates the minimum detectable level, and any points on this line indicate that levels were below the detect- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/12/2947/2002115/0362-028x-69_12_2947.pdf by guest on 27 September 2021 able limit. ⅷ, Control; ⅜, Surfynol; Ⅵ, carvacrol (0.3%); ᭡, carvacrol (0.5%); ᭞, carvacrol (0.7%).

growth of four E. coli O157:H7 strains (H1730, E0019, inhibited the growth of both microorganisms, although eu- F4546, and 932) in the presence of 0.3, 0.5, and 0.9% eugenol only inhibited L. monocytogenes growth at higher genol loaded micelles varied depending on strain (Fig. 3). concentrations. The antimicrobial activity of eugenol and Micellar-encapsulated eugenol was extremely effective as a carvacrol has been attributed to increased proton and po- biocidal agent against colony biofilms of E. coli O157:H7 tassium permeability of cell membranes (25, 52, 53). As strains H1730, E0019, and 932, because no CFUs were the phytophenols are being solubilized and integrated in the recovered after as little as3hofexposure at all tested membranes of bacteria, it becomes more difficult for the concentrations. In contrast, E. coli O157:H7 strain F4546 bacteria to maintain solute gradients across the cytoplasmic (Fig. 3C) proved to be much more resistant to eugenol, with membrane. Immediate decreases in the concentration of bacterial growth observed using 0.3 and 0.5% eugenol. available ATP after less than 30 min of exposure to phy- However, at the highest concentration tested (0.9%), cell tophenols have been reported (53). Interestingly, depletion numbers of strain F4546 was reduced to nondetectable lev- of the internal ATP pool seems to be solely related to de- els within 24 h. creased ATP synthesis because of interference with the Surface growth of four E. coli O157:H7 strains in functionality of protein complexes responsible for ATP pro- the presence of encapsulated carvacrol. Carvacrol-loaded duction. However, the membrane appears to remain imper- micelles were the most effective system tested against E. meable to large molecular weight compounds. In the con- coli O157:H7 (Fig. 4). The application of micellar-encap- text of our study with micellar-encapsulated phytophenols, sulated carvacrol (0.3, 0.5, and 0.7%) led to complete de- surfactants could possibly aid in permeabilization of the struction of E. coli O157:H7, i.e., viable cell numbers fell membrane. Fluorescence studies using planktonic cultures below detectable levels within3hofexposure to the sys- are currently underway in our laboratories to determine tem. whether both antimicrobials and surfactants are found in the membranes. However, results of these studies may be DISCUSSION difficult to translate to more complex biofilms, because This study assessed the antimicrobial effect of surfac- compounds can be expected to interact not only with the tant micelles loaded with two essential oils, eugenol and bacterial cells but also with exopolymeric substances of carvacrol, on the surface growth of two foodborne patho- biofilms. Ultimately, the location and spatial distribution of gens (E. coli O157:H7 and L. monocytogenes) in both ex- both antimicrobials and surfactants within the biofilm need ponential (0, 3, 6, 9, and 24 h of incubation) and stationary to be determined. (48 and 72 h of incubation) phases. Both antimicrobials In our colony biofilm model study, E. coli O157:H7 J. Food Prot., Vol. 69, No. 12 INHIBITION OF L. MONOCYTOGENES AND E. COLI O157:H7 COLONY BIOFILMS 2951

FIGURE 3. Levels (log N/N0)ofE. coli O157:H7 (A) H1730, (B) E0019, (C) F4546, and (D) 932 in the presence of 5% Surfynol 485W and 0, 0.3, 0.5, and 0.9% eugenol as a function of incubation time at 0, 3, 6, 9, 24, 48, and 72 h. Control levels represent growth on tryptic soy agar, and N0 was the level (CFU per membrane) of the control at T ϭ 0. The center dotted line indicates the initial level of bacterial cells (N0). The lower line indicates the minimum detectable level, and any points on this line indicate that levels were below the detect-

able limit. ⅷ, Control; ⅜, Surfynol; Ⅵ, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/12/2947/2002115/0362-028x-69_12_2947.pdf by guest on 27 September 2021 eugenol (0.3%); ᭡, eugenol (0.5%); ᭞, eugenol (0.9%).

FIGURE 4. Levels (log N/N0)ofE. coli O157:H7 (A) H1730, (B) E0019, (C) F4546, and (D) 932 in the presence of 5% Surfynol 485W and 0, 0.3, 0.5, and 0.7% carvacrol as a function of incubation time at 0, 3, 6, 9, 24, 48, and 72 h. Control levels represent growth on tryptic soy agar, and N0 was the level (CFU per membrane) of the control at T ϭ 0. The center dotted line indicates the initial level of bacterial cells (N0). The lower line indicates the minimum detectable level, and any points on this line indicate that levels were below the detectable limit. ⅷ, Con- trol; ⅜, Surfynol; Ⅵ, carvacrol (0.3%); ᭡, carvacrol (0.5%); ᭞, carvacrol (0.7%). 2952 PE´ REZ-CONESA ET AL. J. Food Prot., Vol. 69, No. 12 was more sensitive to eugenol and carvacrol than L. mono- 11 essential oil constituents were tested, carvacrol was the cytogenes. Reported susceptibilities of gram-positive and most potent inhibitor against five foodborne pathogens in- gram-negative organisms against essential oils and their cluding E. coli O157:H7 and L. monocytogenes (27). The compounds vary. Although most studies reported that high antimicrobial activity of carvacrol has been closely gram-positive bacteria were more susceptible to treatment linked to the position and presence of the hydroxyl group with essential oils and their compounds than gram-negative in the phenolic structure as evidenced by the loss of activity bacteria, some contradictory findings have been published in hydroxyl-void monoterpene cyclic hydrocarbons such as (14, 24, 27, 31, 50). For example, Salmonella Enteritidis p-cymene (14). Moreover, the increased length of the alkyl was more susceptible to treatment with steam distillation- chain attached to the phenol ring of eugenol alters the par- extracted mint oil than L. monocytogenes (50). Similarly, titioning coefficient between the solvent and bacterial phas- Vibrio vulnificus was more sensitive to 11 tested essential es directly affecting its antimicrobial activity. oil compounds, including carvacrol, eugenol, geraniol, and The application of both antimicrobial-surfactant com- linalool, than L. monocytogenes (27). It should be noted, binations at sufficiently high concentrations (0.5 to 0.9%) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/12/2947/2002115/0362-028x-69_12_2947.pdf by guest on 27 September 2021 however, that the methods used to apply antimicrobials var- caused destruction of tested strains at Ͼ3 log. A similar log ied. Authors choose different ways to overcome the low reduction (Ͼ4 log) was found in L. monocytogenes strains water solubility of the essential oil compounds. For exam- Scott A and 310 when treated with carvacrol (Fig. 2A and ple, the essential oil compounds were dispersed in ethanol- 2D). Typically, a 3- to 5-log reduction of microbial popu- water mixtures, directly added to the agar, or mixed with a lations is desirable. For example, the AOAC Germicidal small amount of surfactant (e.g., Tween 20) before being and Detergent Sanitizer Test has set a 5-log reduction in added to the agar (14, 24, 27, 31, 50). Variability in the test planktonic microbial populations as a target for approval of protocols highlights the difficulties associated with testing a sanitizer (3). For surface-grown bacteria, a 3-log reduc- antimicrobial compounds that are difficult to disperse in tion (99.9%) after a 0.5- to 5-min treatment has been sug- microbiological model media. Generally, without the aid of gested as a goal for reducing the number of surface attached solubilizing compounds (e.g., surfactants, short chain al- bacteria (1, 18, 33). A dose-related response was observed cohols, etc.), a homogeneous distribution of the compounds when using surfactant-encapsulated carvacrol and eugenol in the test media may be difficult to achieve due to physi- to inhibit and inactivate colony biofilms, especially E. coli cochemical instability mechanisms such as thermodynamic O157:H7 strain F4546 and L. monocytogenes strains Scott incompatibilities that may lead to phase separation of the A and 108. Other authors also reported that minimum levels antimicrobial compound. of polyphenolics are required to significantly affect bacte- We previously reported that the addition of 0.2% eu- rial growth and survival (27, 28). Below these critical lev- genol completely inhibited the growth of both bacteria els, bacterial cells appear to be able to maintain homeosta- when grown as planktonic cultures (22, 23). In colony bio- sis. Bacteria grown in the presence of phytophenols below films, all E. coli O157:H7 strains were inhibited with the minimum inhibitory levels are in fact able to react to Ն0.3% eugenol; however, 0.9% eugenol was needed to in- the superimposed stress by altering their membrane com- hibit all four strains of L. monocytogenes. This is in agree- position, thereby decreasing membrane solubility of phy- ment with observations that bacteria growing on surfaces tophenols (10). However, above critical levels, phytophen- in biofilms are more resistant to antimicrobials than their ols are solubilized in bacterial membranes due to partition- planktonic counterparts (6, 7, 37, 39). Schwach and Zottola ing regardless of the altered membrane composition, there- (39) first reported that treatment of Pseudomonas fragi, Sal- by increasing cell membrane permeability. Eugenol and monella Montevideo, and Bacillus cereus with sodium hy- carvacrol are generally active against a wide selection of pochloride followed by rinsing with water was not effective gram-positive and gram-negative bacteria despite their rel- in completely removing these bacteria from food processing atively low water solubility (14). However, when tested in surfaces. It has been suggested that the physical properties a more complex, multicomponent system, antimicrobial ac- of the surface-grown colony matrix limit the rate of trans- tivity typically decreases due to interaction with these com- port and activity of compounds (7, 44–48). The transport ponents, particularly if their molecular nature is more sim- of active agents from the delivery phase (typically the sur- ilar to that of the phytophenols (41, 51). For example, the rounding aqueous phase) through the matrix to the micro- mainly hydrophobic nature of eugenol and carvacrol can bial cell layer directly attached to the adhering interface lead to rapid partitioning into fat or oil phases. Presence of might be reduced due to physicochemical interactions of the hydroxyl group simultaneously imparts some hydro- disinfectants with organic material or microorganisms in philicity causing the compound to be preferentially located the top layer of the biofilm matrix. Thus, treatment of bio- at the surface of oil phases where they may further interact films with various sanitizers such as monochloramine and with proteins (21). Partitioning into fat and oil phases de- aminotri(methylene-phosphonic acid) pentasodium salt, creases the effective antimicrobial concentration in the may or may not kill the microorganisms. aqueous phase, which subsequently leads to a reduction in Results from this study showed that carvacrol had the observed activity (22, 23). Results of this study suggest higher antimicrobial efficacies than eugenol against the that surfactant-encapsulated eugenol and carvacrol are very eight strains of E. coli O157:H7 and L. monocytogenes test- effective against bacterial surface growth, possibly because ed. A similar finding was recently reported with E. coli (14, the surfactant-based capsule increases the concentration of 35) and E. coli O157:H7 (20). In another study, in which phytophenols near the bacterial membrane despite the po- J. Food Prot., Vol. 69, No. 12 INHIBITION OF L. MONOCYTOGENES AND E. COLI O157:H7 COLONY BIOFILMS 2953 tential compositional complexity of the surface-grown cul- T. J. Montville (ed.), Food microbiology: fundamentals and frontiers. ture. ASM Press, Washington, D.C. 10. Davidson, P. M., and M. A. Harrison. 2002. Resistance and adap- The systems used in this study are thermodynamically tation to food antimicrobials, sanitizers, and other process controls. stable, and, in contrast to emulsions, their size and com- Food Technol. 56:69–78. position does not change over time. Experiments in our 11. Dewanti, R., and A. C. L. Wong. 1995. Influence of culture condi- laboratory have demonstrated their stability over more than tions on biofilm formation by Escherichia coli O157:H7. Int. J. Food 2 years (at room temperature). Efficacies against planktonic Microbiol. 26:147–164. cultures such as E. coli O157:H7 and L. monocytogenes 12. Djordjevic, D., M. Wiedmann, and L. A. McLandsborough. 2002. 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Food Prot. 67:456–462. and biodegradable) surfactants in food preservation is an 18. Frank, J. F., and R. A. N. Chmielewski. 1997. Effectiveness of san- interesting alternative to traditional preservation systems. itation with quaternary ammonium compound or chlorine on stain- Nevertheless, the use of large quantities of essential oil less steel and other domestic food-preparation surface. J. Food Prot. 60:43–47. compounds may impart flavors that could limit their use in 19. Frank, J. F., and R. A. Koffi. 1990. Surface-adherent growth of Lis- certain products (54). teria monocytogenes is associated with increased resistance to surfactant sanitizers and heat. J. Food Prot. 53:550–554. ACKNOWLEDGMENTS 20. Friedman, M., P. R. Henika, and R. E. Mandrell. 2002. 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