Carvacrol and Cinnamaldehyde Inactivate Antibiotic-Resistant <I

234

Journal of Food Protection, Vol. 73, No. 2, 2010, Pages 234–240

Carvacrol and Cinnamaldehyde Inactivate Antibiotic-Resistant Salmonella enterica in Buffer and on Celery and Oysters

SADHANA RAVISHANKAR,1* LIBIN ZHU,1 JAVIER REYNA-GRANADOS,1 BIBIANA LAW,1 LYNN JOENS,1 AND MENDEL FRIEDMAN2

1Department of Veterinary Science and Microbiology, University of Arizona, 1117 East Lowell Street, Tucson, Arizona 85721; and 2U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Produce Safety and Microbiology Research, 800 Buchanan Street, Albany, California 94710, USA Downloaded from http://meridian.allenpress.com/jfp/article-pdf/73/2/234/1679919/0362-028x-73_2_234.pdf by guest on 27 September 2021 MS 09-228: Received 20 May 2009/Accepted 25 September 2009

ABSTRACT The emergence of antibiotic-resistant Salmonella is of concern to food processors. The objective of this research was to identify antimicrobial activities of cinnamaldehyde and carvacrol against antibiotic-resistant Salmonella enterica in phosphate- buffered saline (PBS) and on celery and oysters. Twenty-three isolates were screened for resistance to seven antibiotics. Two resistant and two susceptible strains were chosen for the study. S. enterica cultures (105 CFU/ml) were added to different concentrations of cinnamaldehyde and carvacrol (0.1, 0.2, 0.3, and 0.4% [vol/vol]) in PBS, mixed, and incubated at 37uC. Samples were taken at 0, 1, 5, and 24 h for enumeration. Celery and oysters were inoculated with S. enterica (106–7 CFU/ml), treated with 1% cinnamaldehyde or 1% carvacrol, incubated at 4uC, and then sampled for enumeration on days 0 and 3. Both antimicrobials induced complete inactivation of S. enterica in PBS at 0.3 and 0.4% on exposure, and on 0.2% in 1 h. Exposure to cinnamaldehyde at 0.1% inactivated all pathogens at 1 h, and survivors were observed only for Salmonella Newport with 0.1% carvacrol at 1 h. In celery, 1% carvacrol reduced S. enterica populations to below detection on day 0, while 1% cinnamaldehyde reduced populations by 1 and 2.3 log on day 0 and day 3, respectively. In oysters, both antimicrobials caused about 5-log reductions on day 3. These results show the potential antimicrobial effects of carvacrol and cinnamaldehyde against antibiotic- resistant S. enterica in vitro and in foods.

Salmonella enterica is one of the leading causes of contaminated dairy plant was implicated in an outbreak foodborne illnesses. According to the 2006 preliminary involving 180,000 cases associated with Salmonella Typhi- FoodNet data with estimates from the Center for Disease murium that was resistant to five antibiotics (38). Previous- Control and Prevention, of the total 17,252 laboratory- ly, Niemira et al. (29), Rajashekara et al. (34), and confirmed cases of foodborne infections in 10 states, 6,655 Schwaiger et al. (40) reported on antibiotic-resistant S. (overall incidence of 14.81 per 10,000 people) were due to enterica isolates that are relevant to the present study. S. enterica (45, 46). There is therefore, an urgent need to Salmonella-resistant strains were also found to be devise appropriate control measures for this pathogen. prevalent in a large number of imported seafood samples Antibiotic-resistant strains of S. enterica have emerged (32, 48). The majority of worldwide outbreaks associated in recent years and are a concern to the food industry. with bivalve shellfish have been linked to oysters, and then Antibiotic resistance may arise from several distinct to clams and mussels. Oysters, which are filter feeders, often pathways: changes in permeability in the bacterial cell wall, concentrate pathogenic bacteria from marine and brackish which restricts access of antibiotics to target sites; efflux of waters (8). Rising ocean temperatures seem to contribute to the antibiotic from the cell; and formation of new metabolic observed increases in outbreaks associated with oysters pathways for survival to overcome those inhibited by the (27). Studies have shown that Salmonella was present in antibiotic. A common mechanism is the acquisition of 7.4% of U.S. market oysters (2, 3). These oyster isolates external genes that provide resistance to an entire class of were susceptible to ciprofloxacin, gentamycin, and trimeth- antibiotics. Bacteria also possess pumps that export oprim-sulfamethoxazole, but were largely resistant to antibiotics before they can reach sensitive intracellular ampicillin and tetracycline. Successful efforts to inactivate targets. The use of antibiotics in animal husbandry and S. enterica on oysters include the use of high pressure (26) human medicines creates selective pressure that favors the and ionizing radiation (22). Chitosan treatment proved development of selective resistance among microorganisms. ineffective over a 12-day storage period (7). Extensive use of antibiotics in humans, animals, and plants A U.S. Food and Drug Administration survey of high- has resulted in formation of a pool of resistance genes in the volume imported fresh produce including celery showed that a environment (10, 39). For example, pasteurized milk from a significant fraction of all vegetables were contaminated with Salmonella and other pathogens (4). Quiroz-Santiago et al. * Author for correspondence. Tel: 520-626-1499; Fax: 520-621-6366; (33) reported that Salmonella contamination of vegetables E-mail: [email protected]. imported from Mexico ranged as follows (percentage of J. Food Prot., Vol. 73, No. 2 INACTIVATION OF ANTIBIOTIC-RESISTANT SALMONELLA 235 isolates from all samples): parsley (12%); cilantro (11%); washed twice in sterile buffered peptone water (BPW; Difco, broccoli and cauliflower (9%); long lettuce, spinach, and Becton Dickinson). Cells suspended in BPW to a concentration of 9 watercress (7%); parsley (6%); beets (4%); celery and about 10 CFU/ml (from overnight culture) were then diluted in Romaine lettuce (3%); and cabbage and potatoes (1%). BPW to the required concentration, depending on the initial These considerations point to the need to develop new inoculum needed for experiments. Viable organisms after each treatment were enumerated by serially diluting in BPW and plating alternatives for standard antibiotics that can be effective on xylose-lysine-desoxycholate agar (XLD; Difco, Becton Dick- against antibiotic-resistant bacteria. Compounds derived from inson) and tryptic soy agar (TSA; Difco, Becton Dickinson), and plants such as essential oils, their active components, spices, then incubating the cells at 37uC for 24 to 48 h. For all studies and plant extracts have shown antimicrobial effects against a involving plant antimicrobials, controls not treated with these number of foodborne pathogens. For example, carvacrol, antimicrobials were used as standards for comparison to those thymol, and cinnamic acid were found to be effective at samples treated with antimicrobials. concentrations of 1 to 7.5 mmol21 against Salmonella Typhimurium and Escherichia coli (18, 30).Previously,we Screening of S. enterica isolates for antibiotic resistance. To found that carvacrol also inhibited growth of E. coli O157:H7 determine antibiotic resistance, Salmonella strains were screened Downloaded from http://meridian.allenpress.com/jfp/article-pdf/73/2/234/1679919/0362-028x-73_2_234.pdf by guest on 27 September 2021 and the formation of heterocyclic amines in grilled beef patties with BD BBL Sensi-Disc antibiotics (BBL, Becton, Dickinson, (20), and that carvacrol and cinnamaldehyde in edible apple Sparks, MD). The antibiotics used from the Sensi-Disc were amoxicillin–clavulanic acid (20–10 mg), ampicillin (10 mg), cefoxitin films inhibited the growth of E. coli O157:H7 and S. enterica (30 mg), chloramphenicol (30 mg), streptomycin (10 mg), trimetho- on poultry, and Listeria monocytogenes on ham surfaces (36). prim-sulfamethoxazole (1.25–23.75 mg), and tetracycline (30 mg). A Citrus products and extracts have been found to be effective breakpoint approach was used to determine resistance of S. enterica against E. coli O157:H7 and Salmonella Typhimurium (6, isolates to the various antibiotics tested. The breakpoints used for the 28). Pastes of ginger and garlic inhibited E. coli O157:H7 and control strain E. coli ATCC 25922 correspond to values established Salmonella Typhimurium (21, 42). Other natural antimicro- for Enterobacteriaceae by the Clinical Laboratory Standards Institute bials include tea catechins, phenolic acids, and aldehydes (12, and approved by the U.S. Food and Drug Administration. The 13, 15–17, 19). Limited studies have been conducted to find following observed breakpoints were used to define resistance of out whether these compounds are effective against antibiotic- the bacteria to the seven antibiotics: amoxicillin–clavulanic acid resistant bacteria (13, 14, 35). (#13 mm), ampicillin (#13 mm), cefoxitin (#14 mm), chloram- The objective of the present study was to identify phenicol (#12 mm), streptomycin (#11 mm), trimethoprim-sulfa- effective plant-derived antimicrobials against antibiotic- methoxazole (#10 mm), and tetracycline (#14 mm). resistant S. enterica in phosphate-buffered saline (PBS) Isolates were grown on Luria-Bertani agar (Difco, Becton Dickinson) plates, and CFUs were suspended in 0.85% NaCl to an buffer and on celery and oysters, as model foods for optical density at 600 nm of 0.80 (equivalent to a McFarland vegetables and seafood, respectively. standard of 1.0). The suspension was then swabbed confluently onto Mueller-Hinton agar (Difco, Becton Dickinson) plates. Then, MATERIALS AND METHODS a Sensi-Disc for each antibiotic was placed on the plates. After Bacterial strains and antimicrobials. S. enterica was the incubation at 37uC for 24 to 48 h, the MIC was determined as per test organism used. After antibiotic screening, Salmonella Newport the manufacturer’s instructions. (resistant to six of seven antibiotics tested), Salmonella Typhimur- ium DT104 H3278 (resistant to four of seven antibiotics tested), Antimicrobial activities of cinnamaldehyde and carvacrol Salmonella Typhimurium (susceptible to all seven antibiotics against resistant and susceptible S. enterica in PBS buffer. The tested), and Salmonella Enteritidis ATCC 13076 (susceptible to all antimicrobial compounds (cinnamaldehyde and carvacrol) were seven antibiotics tested) were used for antimicrobial studies. suspended in sterile PBS. A 0.5% stock solution was prepared in Salmonella Typhimurium and Salmonella Typhimurium DT104 PBS. The stock solution was serially diluted in microcentrifuge were provided by Dr. V. K. Juneja, U.S. Department of Agriculture, tubes to 0.4, 0.3, 0.2, and 0.1% to a volume of 90 ml. The test Agricultural Research Service, Eastern Regional Research Center organism (10 ml, 105 CFU/ml) was then added to each micro- (Wyndmoor, PA). All Salmonella isolates were first screened for their centrifuge tube, mixed thoroughly, and sampled immediately (0- antibiotic resistance, as described below. The results of the screen min sample). The 0-min sample was spread plated on XLD. The showed that Salmonella Newport and Salmonella Typhimurium DT samples were then incubated at 37uC. After incubation, the 104 were antibiotic resistant, and Salmonella Typhimurium and samples were evaluated at 0, 1, 5, and 24 h. At each sampling, Salmonella Enteritidis were susceptible. serial dilutions were done in BPW and plating on XLD. CFUs were Two antimicrobial compounds obtained from Sigma (St. counted after incubation of the plates at 37uC for 24 h. Louis, MO) were evaluated for effectiveness against both resistant and susceptible strains, trans-cinnamaldehyde (.99.5% pure, Antimicrobial activities of cinnamaldehyde and carvacrol molecular weight 132.2, Chemical Abstracts Services [CAS] no. against resistant and susceptible S. enterica on celery. Fresh 14371-10-9) and carvacrol (.98% pure, molecular weight 150.2, celery was obtained from local retail outlets. Celery was washed CAS no. 499-75-2). thoroughly in deionized water and then cut into 10-g pieces. The cut pieces were exposed to UV light under a biohood for 30 min. Bacterial culture preparation and media. Each Salmonella Celery samples were then dip inoculated in Salmonella Newport culture was prepared by inoculating cryopreserved cells in tryptic culture (10627 CFU/ml) for 2 min, and then dried for 1 h under a soy broth (Difco, Becton Dickinson, Sparks, MD) and then biohood. The inoculated celery samples were immersed in 1% incubating the cells overnight (18 to 20 h) at 37uC. Two transfers cinnamaldehyde or 1% carvacrol in PBS for 10 min. After were done before a working culture was prepared. Cells (109 CFU/ treatment, celery samples were stored in sterile petri dishes at ml) were harvested by centrifugation (2,000 | g for 10 min) and 4uC for 3 days. Enumerations of surviving bacteria were done on 236 RAVISHANKAR ET AL. J. Food Prot., Vol. 73, No. 2

TABLE 1. Inhibition-zone diameter values of Salmonella enterica isolates for selected antibioticsa

Antibiotic (mg):

Amoxicillin– Trimethoprim- clavulanic acid Ampicillin Cefoxitin Chloramphenicol Streptomycin sulfamethoxazole Tetracycline Salmonella strains (20/10) (10) (30) (30) (10) (1.25–23.75) (30)

b ST DT104 H3278 16.7 0.0 25 0.0 8.3 21.7 10.3 Muenchen 25.4 23.2 24.8 27.2 16.1 27.4 19.5 ST 848 28.7 26.7 26 29 15.1 27.2 20.7 ST DT104 H3880 17.5 0.0 24.1 0.0 7.6 22.7 9.5 ST DT104 H2662 14.3 0.0 25 0.0 8.5 22.3 9.5 SE 13026c 27.3 25.8 28.1 26.7 20 31.3 20.6 SE H3502 25.5 23.6 24.6 24.7 20.6 26.8 18.7 SE H3527 25.9 25.3 24.8 25.2 19.1 26.8 21.2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/73/2/234/1679919/0362-028x-73_2_234.pdf by guest on 27 September 2021 S9d 28 25 26.3 27.6 13.6 24.1 0.0 S19d 20 0.0 21.4 23.1 14.5 25 0.0 S20d 27 24 25.8 27.2 15.2 28.2 18.7 ST 28.2 29.7 29.3 28.6 20.5 28.8 24 ST 645 19.1 0.0 26.7 24.2 14.9 22 19 Newport 9.9 0.0 9.5 0.0 0.0 21.4 0.0 Cubana 25.6 24.3 24.3 25 18.8 26.9 19.3 ST DT104 H3402 16.2 0.0 23.6 26.7 8.5 21.9 9 Enteritidis 26.6 23.3 22.7 25.6 16 25.2 21.2 ST DT104 H3380 15 0.0 25.4 0.0 7.3 22.6 9 Saint Paul 26.5 23.6 25 25.1 14 25.2 20.2 SE 13076 27.7 26.5 27 26.4 17.8 31 21 Tennessee 25 23.1 22.9 23.2 12.8 25.1 17 Choleraesuis 28.2 25 27.4 26.7 16.8 33.1 21.7 Havana 25.1 23.8 22.5 22.7 17 25.3 18.2 E. coli e 21.4 18.8 26.6 25.4 16 25.3 21.2 a Resistance breakpoints used were as follows, and are expressed in millimeters (0.0 mm ¡ 0.05): amoxicillin–clavulanic acid # 13, ampicillin # 13, cefoxitin # 14, chloramphenicol # 12, streptomycin # 11, trimethoprim-sulfamethoxazole # 10, and tetracycline # 14. Values in boldface type are resistance values. b ST, Salmonella Typhimurium isolate. c SE, Salmonella Enteritidis isolate. d Copper-resistant Salmonella isolate. e The E. coli strain used was ATCC 25922. day 0 and day 3. Samples were stomached in 90 ml of BPW by RESULTS AND DISCUSSION using a stomacher (Seward, Ltd., London, UK) at normal speed for 1 min, serially diluted in BPW, and then plated on XLD and TSA. Screening of S. enterica isolates for antibiotic resis- Colonies were counted after 24 h of incubation at 37uC. Untreated tance. A total of 23 isolates were tested for susceptibility to inoculated and noninoculated celery samples served as controls. amoxicillin–clavulanic acid, ampicillin, cefoxitin, chloramphenicol, streptomycin, trimethoprim-sulfamethoxazole, and Antimicrobial activities of cinnamaldehyde and carvacrol tetracycline (Table 1). Of the 23 isolates, 9 (39.1%) were against resistant and susceptible S. enterica on oysters. Oysters resistant to at least one antibiotic, and 14 isolates (60.9%) were (in shell) were purchased from local grocery stores. They were susceptible to all seven antibiotics tested. Only one isolate shucked and washed 10 times in deionized water, and were then kept immersed in deionized water overnight at 4uC. The cleaned oysters (4.3%) was resistant to amoxicillin–clavulanic acid, 8 (34.8%) were cut into 10-g pieces, dipped in hot water for 40 s, and dried were resistant to ampicillin and tetracycline, 6 (26.1%) were under a biohood for 30 min. Oyster samples were dip inoculated in resistant to streptomycin, 5 (21.7%) to chloramphenicol, and 1 Salmonella Newport culture (10627 CFU/ml) for 2 min, and then isolate (4.3%) to amoxicillin–clavulanic acid or to cefoxitin. dried for 1 h under a biohood. Inoculated samples were then None of the tested isolates was resistant to trimethoprim- immersed in 1% cinnamaldehyde or 1% carvacrol in PBS for 10 min sulfamethoxazole. These results suggest that the latter or 1 h. All samples were stored in sterile petri dishes at 4uCfor3 combination has the potential to be a drug of choice for days. Enumerations of surviving bacteria were done on day 0 and day treating salmonellosis in animals and humans. 3, according to procedures described above for celery. Untreated Salmonella Newport was found to be the most resistant inoculated and noninoculated oyster samples served as controls. serotype, showing resistance to six of the seven antibiotics Statistical analysis. Each experiment was repeated at least tested. Four strains of Salmonella Typhimurium DT104 three times. Tables 2 through 4 show means and standard deviations (H3278, H3880, H2662, and H3380) were each resistant to (SD) for the surviving bacterial populations for each sampled time. four antibiotics. Salmonella Newport and Salmonella Typhi- J. Food Prot., Vol. 73, No. 2 INACTIVATION OF ANTIBIOTIC-RESISTANT SALMONELLA 237

TABLE 2. Antimicrobial effects of carvacrol and cinnamaldehyde against antibiotic-resistant and -susceptible Salmonella enterica isolatesa

Antimicrobial concn (%)b:

0 0.1 0.2 0.3 Sampling time Salmonella isolate (h) Control CAR CIN CAR CIN CAR CIN

c Typhimurium 0 5.0 ¡ 0.1 3.1 ¡ 0.7 4.0 ¡ 0.1 NG NG NG NG 1 5.1 ¡ 0.0 NG NG NG NG NG NG 5 5.1 ¡ 0.0 NG NG NG NG NG NG 24 4.5 ¡ 0.5 NG NG NG NG NG NG Typhimurium 0 5.0 ¡ 0.2 NG 4.8 ¡ 0.0 NG 4.8 ¡ 0.1 NG NG H3278 1 5.0 ¡ 0.1 NG NG NG NG NG NG 5 7.0 ¡ 0.2 NG NG NG NG NG NG Downloaded from http://meridian.allenpress.com/jfp/article-pdf/73/2/234/1679919/0362-028x-73_2_234.pdf by guest on 27 September 2021 24 8.0 ¡ 0.2 NG NG NG NG NG NG Enteritidis 0 5.1 ¡ 0.0 4.0 ¡ 0.2 4.3 ¡ 0.0 NG 3.9 ¡ 0.1 NG NG 1 5.1 ¡ 0.1 NG NG NG NG NG NG 5 6.8 ¡ 0.4 NG NG NG NG NG NG 24 8.7 ¡ 0.1 NG NG NG NG NG NG Newport 0 4.9 ¡ 0.0 3.9 ¡ 0.1 4.2 ¡ 0.1 NG NG NG NG 1 5.8 ¡ 0.1 3.0 ¡ 0.3 NG NG NG NG NG 5 6.9 ¡ 0.0 NG NG NG NG NG NG 24 7.7 ¡ 0.6 NG NG NG NG NG NG a Values are means ¡ SD (n ~ 3) and are expressed in log CFU per milliliter. b CAR, carvacrol; CIN, cinnamaldehyde. c NG, no growth observed. murium DT104 H3278 were selected as the resistant strains (Table 2). Both compounds at concentrations of 0.4 and for the present study on effects of antimicrobials in vitro. 0.3% completely inactivated both resistant and sensitive Salmonella Typhimurium DT104 strain H3402 exhibited organisms at all sampling points. Because no survivors were resistance toward three antibiotics, and copper-resistant strain detected at all times at 0.4%, the data for this concentration S 19 toward two antibiotics. Copper-resistant strain S 9 and are not included in Table 2. With 0.2% carvacrol, no Salmonella Typhimurium 645 were each resistant to one survivors were detected at all times for all four isolates. antibiotic. Of the 14 strains susceptible to all seven antibiotics, With 0.2% cinnamaldehyde, no survivors were detected at two strains (Salmonella Typhimurium and Salmonella all times for Salmonella Typhimurium and Salmonella Enteritidis) were also evaluated for their resistance or Newport. For Salmonella Typhimurium DT104 H3278, susceptibility to the plant compounds. However, the intent there was very limited reduction in the population at 0 h, of the study was not to determine whether antibiotic-resistant and no survivors were detected thereafter at $1 h with and antibiotic-sensitive bacteria would exhibit differences in 0.2% cinnamaldehyde. With 0.2% cinnamaldehyde, for susceptibility to the plant compounds. Salmonella Enteritidis, the population declined by about 1 log at 0 h, and no survivors were detected thereafter. Antimicrobial activities of cinnamaldehyde and Carvacrol at 0.1% reduced the population of Salmonella carvacrol against S. enterica in PBS buffer. Cinnamal- Typhimurium by about 2 log, and that of Salmonella dehyde and carvacrol were tested at 0.1, 0.2, 0.3, and 0.4% Enteritidis and Salmonella Newport by about 1 log each, concentrations in PBS against S. enterica at 0, 1, 5, and 24 h immediately on exposure. No survivors were detected at 1 h or later for Salmonella Typhimurium and Salmonella Enteritidis. TABLE 3. Survival of antibiotic-resistant Salmonella Newport on For Salmonella Newport, a 2.8-log reduction in population celery treated with carvacrol and cinnamaldehydea was observed at 1 h, and no survivors were detected thereafter. With 0.1% carvacrol, no survivors were detected at all times Agar for Salmonella Typhimurium DT104 H3278. Exposure to Treatmentb medium Day 0 Day 3 cinnamaldehyde at 0.1% resulted in inactivation of all Control TSA 4.9 ¡ 0.2 4.6 ¡ 0.3 organisms at 1 h or longer for all four isolates tested. XLD 4.9 ¡ 0.1 4.4 ¡ 0.3 Immediately on exposure, the reduction was 1 log for 1% CAR TSA ,1.0 ¡ 0.0 ,1.0 ¡ 0.0 Salmonella Typhimurium, slightly ,1 log for Salmonella XLD ,1.0 ¡ 0.0 ,1.0 ¡ 0.0 Enteritidis and Salmonella Newport, and there was no ¡ ¡ 1% CIN TSA 3.8 0.1 2.3 0.3 reduction for Salmonella Typhimurium DT104 H3278. ¡ ¡ XLD 3.8 0.1 2.2 0.3 When comparing the two antimicrobials, carvacrol a Values are means ¡ SD and are expressed in log CFU per gram. showed stronger activity than did cinnamaldehyde at low b CAR, carvacrol; CIN, cinnamaldehyde. concentrations (0.1 to 0.2%) against S. enterica. 238 RAVISHANKAR ET AL. J. Food Prot., Vol. 73, No. 2

TABLE 4. Survival of antibiotic-resistant Salmonella Newport on against both susceptible and resistant pathogens, we briefly oysters treated with carvacrol and cinnamaldehydea present published information on the mechanism of the Treatmentb Time Agar Day 0 Day 3 antimicrobial effects of carvacrol and cinnamaldehyde (5, 11). Ultee et al. (43) showed that the consequences of Control 10 min TSA 5.7 ¡ 0.1 5.6 ¡ 0.2 exposing Bacillus cereus to carvacrol include depletion of XLD 5.7 ¡ 0.1 5.1 ¡ 0.1 the intracellular ATP pool, change in membrane potential, 1 h TSA 5.7 ¡ 0.2 5.5 ¡ 0.1 and increase in permeability of the cytoplasmic membrane ¡ ¡ XLD 5.8 0.1 5.0 0.1 to proteins and potassium ions. Other studies showed that ¡ ¡ CAR 10 min TSA 4.7 0.2 4.0 0.1 carvacrol damaged the outer membrane of Pseudomonas XLD 4.6 ¡ 0.2 3.5 ¡ 0.4 aeruginosa, destroying the lipopolysaccharide barrier (9), 1 h TSA 4.2 ¡ 0.4 ,1.0 ¡ 0.0 XLD 3.9 ¡ 0.6 ,1.0 ¡ 0.0 and that carvacrol can form ion channels though the CIN 10 min TSA 4.9 ¡ 0.1 4.0 ¡ 0.2 membrane, partitioning the fatty acid chains of the XLD 4.8 ¡ 0.0 3.3 ¡ 0.5 phospholipids, thereby permitting ions to leave the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/73/2/234/1679919/0362-028x-73_2_234.pdf by guest on 27 September 2021 1 h TSA 4.6 ¡ 0.4 ,1.4 ¡ 0.74 cytoplasm (43). Leakage of phosphate ions occurred in XLD 4.3 ¡ 0.3 ,1.0 ¡ 0.6 Staphylococcus aureus and P. aeruginosa cells treated with oregano essential oil, which has carvacrol as a main a Values are means ¡ SD and are expressed in log CFU per gram. antimicrobial component (13, 24). b CAR, carvacrol; CIN, cinnamaldehyde. Friedman (11) describes the use of autofluorescence spectra to determine the effect of carvacrol on E. coli C600. Antimicrobial activities of cinnamaldehyde and The data showed significant changes at much lower carvacrol against S. enterica on celery. Fresh produce concentrations of carvacrol (0.01 mM) than changes in including celery can be a vehicle for transmission of S. membrane potential or release of ATP (ATP flux) after enterica and other pathogens (1, 4). With the possible disruption of the bacterial cell membrane, occurring at higher exception of gamma irradiation (25), to our knowledge, no concentrations of carvacrol (1 to 2 mM). This observation effective treatments are available against antibiotic-resistant suggests that autofluorescence detects physiological responses S. enterica on celery. to lower concentrations of carvacrol more efficiently than it The results of the present study listed in Table 3 show does changes in membrane potential or release of ATP that dipping of celery samples in 1% carvacrol for 10 min occurring at higher concentrations of carvacrol. induced complete inactivation of Salmonella Newport on Disintegration of bacterial outer membrane has not day 0 and day 3, respectively. By contrast, celery samples been observed with cinnamaldehyde. Instead, cinnamalde- dipped in 1% cinnamaldehyde showed only 1- and 2.3-log hyde is thought to interfere with the activity of some reductions on day 0 and day 3, respectively. In general, enzymes. For example, the carbonyl group of cinnamalde- there was no difference between counts of surviving hyde appears to bind to proteins in Enterobacter aerogenes, salmonellae on selective (XLD) and nonselective (TSA) preventing the action of amino acid carboxylases (47). media (Table 3), indicating there was no substantial injury Comparison of the antibiotic-resistant versus -sensitive caused to the bacterial cells on celery due to antimicrobial isolates of S. enterica to carvacrol and cinnamaldehyde treatments. observed in the present study does not allow specific These results show that the effectiveness of carvacrol conclusions about mechanisms. It is, however, noteworthy against antibiotic-resistant Salmonella Newport on celery is that Salmonella Newport, which was found to be resistant to greater than that of cinnamaldehyde. six of the seven antibiotics tested, showed different susceptibilities to carvacrol and cinnamaldehyde than the Antimicrobial activities of cinnamaldehyde and other strains demonstrated. For example, no Salmonella carvacrol against S. enterica on oysters. Table 4 shows Newport survivors were detected even at 0 min with the that dipping of oyster samples in 1% carvacrol solution for 0.2% cinnamaldehyde treatment, while survivors were 10 min resulted in a reduced count of Salmonella by about 1 detected for the other resistant isolate Salmonella Typhi- and 1.5 log on days 0 and 3, respectively. Dipping the murium DT104 H3278 and susceptible isolate Salmonella oysters in 1% carvacrol for 1 h showed about 1.5- and 5-log Enteritidis. With 0.1% carvacrol, no survivors were detected reductions (no survivors detected) at day 0 and day 3, for Salmonella Typhimurium DT104 H3278 soon after respectively. Dipping treatments in 1% cinnamaldehyde exposure, while survivors were detected in case of showed about similar results. The counts on selective (XLD) Salmonella Newport up to 1 h, and in case of the other and nonselective (TSA) media were similar (Table 4), two susceptible isolates, up to 0 h. However, at .0.2% indicating no significant injury to S. enterica cells due to concentration, both antimicrobials completely inactivated antimicrobial treatments on oysters. the antibiotic-resistant and -susceptible isolates of S. These results show that both carvacrol and cinnamal- enterica. Our results suggest that the various S. enterica dehyde are effective natural antimicrobials against antibiot- isolates may not be equally susceptible to inactivation, and ic-resistant Salmonella Newport on oysters. that the antimicrobials may act by similar mechanisms against resistant and susceptible S. enterica isolates. Mechanism of antimicrobial effects. To facilitate Related relevant studies showed that (i) the essential oil understanding the effectiveness of plant antimicrobials of Origanum vulgare exhibited high antimicrobial activity J. Food Prot., Vol. 73, No. 2 INACTIVATION OF ANTIBIOTIC-RESISTANT SALMONELLA 239 against S. enterica at ,1% (31); (ii) cinnamaldehyde, 10. Doyle, M. P., F. Busta, B. R. Cords, P. M. Davidson, J. Hawke, H. S. carvacrol, and thymol completely inhibited growth of B. Hurd, R. E. Isaacson, K. Matthews, J. Maurer, J. Meng, T. J. Montville, T. R. Shryock, J. N. Sofos, A. K. Vidaver, and L. Vogel. cereus in carrot broth stored at 16uC for more than 60 days 2006. Antimicrobial resistance: implications for the food system. An (44); (iii) carvacrol prevented biofilm formation of dual- expert report, funded by the IFT Foundation. Compr. Rev. Food Sci. species bacteria including S. aureus and Salmonella Food Saf. 5:71–137. Typhimurium (23); and (iv) drug resistance did not affect 11. Friedman, M. 2006. Antibiotic activities of plant compounds against the heat resistance of Salmonella serotypes in cooked nonresistant and antibiotic-resistant foodborne human pathogens. ground beef (41). ACS Symp. Ser. 961:167–183. 12. Friedman, M. 2007. Overview of antibacterial, antitoxin, antiviral, The results of the present study show that both and antifungal activities of tea compounds. Mol. Nutr. Food Res. 51: carvacrol and cinnamaldehyde inactivated both antibiotic- 116–134. resistant and -susceptible S. enterica isolates in PBS and on 13. Friedman, M., R. Buick, and C. T. Elliott. 2004. Antibacterial two foods, celery and oysters. The antibiotic-resistant activities of naturally occurring compounds against antibiotic- isolates largely exhibited similar susceptibilities to inacti- resistant Bacillus cereus vegetative cells and spores, Escherichia

coli, and Staphylococcus aureus. J. Food Prot. 67:1774–1778. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/73/2/234/1679919/0362-028x-73_2_234.pdf by guest on 27 September 2021 vation as did the susceptible isolates. Whether these 14. Friedman, M., R. Buick, and C. T. Elliott. 2006. Antimicrobial compounds are equally effective against these and other activities of plant compounds against antibiotic-resistant Micrococcus resistant pathogens (such as vibrios in seafood) in other luteus. Int. J. Antimicrob. Agents 28:156–158. contaminated human foods, animal feeds, and infected 15. Friedman, M., P. R. Henika, C. E. Levin, and R. E. Mandrell. 2004. animals and humans merits further investigation. However, Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple we do not know whether the bacteria would develop cross- juice. J. Agric. Food Chem. 52:6042–6048. resistance toward the evaluated plant compounds as they did 16. Friedman, M., P. R. Henika, C. E. Levin, and R. E. Mandrell. 2006. with the synthetic antimicrobials chlorhexidine and quater- Antimicrobial wine formulations against the foodborne pathogens nary ammonium compounds (37). Finally, the results of the Escherichia coli O157:H7 and Salmonella enterica. J. Food Sci. 71: present study suggest that adding carvacrol or cinnamalde- M245–M251. hyde to tanks containing oysters or other seafood may 17. Friedman, M., P. R. Henika, C. E. Levin, R. E. Mandrell, and N. Kozukue. 2006. Antimicrobial activities of tea catechins and inhibit foodborne pathogenic bacteria during storage. theaflavins and tea extracts against Bacillus cereus. J. Food Prot. 69:354–361. ACKNOWLEDGMENT 18. Friedman, M., P. R. Henika, and R. E. Mandrell. 2002. 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