Effect of Subinhibitory Concentrations of Plant-DerivedMolecules in Increasing the Sensitivity of Multidrug-Resistant

Salmonella enterica Serovar Typhimurium DT104to Antibiotics

Anup Kollanoor Johny, Thomas Hoagland, and Kumar Venkitanarayanan

Abstract

This study investigated the efficacy of plant-derived antimicrobials, namely, trans-cinnamaldehyde, b-resorcylicacid, carvacrol, thymol, and eugenol or their combination, in increasing the sensitivity of Salmonella Typhi-murium DT104 to five antibiotics. The subinhibitory concentrations of each antimicrobial or their combinationcontaining concentrations lower than the individual subinhibitory concentrations were added to tryptic soybroth supplemented with antibiotics at their respective break points for resistance. Salmonella TyphimuriumDT104 was inoculated into tryptic soy broth at *6 log CFU=mL, and growth (optical density at 600 nm) wasdetermined before and after incubation at 378C for 24 hours. Appropriate controls were included. Duplicatesamples were assayed and the experiment was replicated three times. Trans-cinnamaldehyde increased thesensitivity of Salmonella Typhimurium DT104 ( p< 0.05) toward all five antibiotics, namely, ampicillin, chlor-amphenicol, streptomycin, sulfamethoxazole, and tetracycline, thereby making the pathogen susceptible todrugs. Thymol made the pathogen susceptible to all four antibiotics except ampicillin, whereas carvacrol in-creased the sensitivity to two antibiotics (chloramphenicol and sulfamethoxazole for strain H3380, and strep-tomycin and sulfamethoxazole for strain 43). The combination of five molecules was more effective thanindividual ones ( p< 0.05) in rendering the pathogen susceptible to the antibiotics. Results indicate that thesenatural molecules individually and synergistically increased the sensitivity of Salmonella Typhimurium DT104 toall the five antibiotics, and justify future studies to control antibiotic resistance of the pathogen in food animalsusing these plant molecules.

Introduction

Salmonella Typhimurium definitive phage-type 104(Salmonella Typhimurium DT104) is one of the multidrug-

resistant clonal groups of Salmonella Typhimurium that posesignificant public health problem worldwide (McEwen andFedorka-Cray, 2002; Varga et al., 2008). Salmonella Typhi-murium DT104 isolates are characterized by resistance to upto nine antibiotics, including ampicillin, chloramphenicol,streptomycin, sulfonamides, and tetracylines (ACSSuT) (VanDuijkeren et al., 2003). Resistance to ACSSuT is attributed tothe presence of five genes, aadA2, sul1, a cmlA homolog, tetA,and blaP1, that confer resistance to streptomycin and specti-nomycin, sulfonamides, chloramphenicol, tetracyclines, andb-lactam antibiotics, respectively (Ridley and Threlfall, 1998;Briggs and Fratamico, 1999). Salmonella Typhimurium DT104is believed to have acquired the resistance plasmids from

aquaculture pathogens, Pasteurella piscicida and Vibrio angu-illarum (Briggs and Fratamico, 1999; Humprey, 2001).

An estimated range of 59,200–296,000 cases of DT104 in-fections occur in the United States accounting for a loss of*$360–$900 million annually (Hogue et al., 1997). The prev-alence of Salmonella Typhimurium DT104 infection has in-creased from a scanty 0.6% of human isolates in 1979 to 23% in2004 (CDC, 2007). A wide range of animals and birds, in-cluding cattle, calves, goats, sheep, pigs, chickens, turkeys,ducks, geese, and game birds, can act as reservoirs of Salmo-nella Typhimurium DT104, with significant incidence ratesnoticed in cattle and pigs (Perron et al., 2007). It is widelyaccepted that Salmonella Typhimurium DT104 strains arespread from cattle to pigs and humans, underscoring itszoonotic transmission and existence in natural environment.Moreover, several common food products such as roast beef,pork sausage, cooked meats, chicken legs, unpasteurized

Department of Animal Science, University of Connecticut, Storrs, Connecticut.

FOODBORNE PATHOGENS AND DISEASEVolume 7, Number 10, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089=fpd.2009.0527

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milk, and cheese serve as potential vehicles. Recently, it hasbeen reported that ampicillin resistance could be transferredfrom Salmonella Typhimurium DT104 strains to other bacteriasuch as Escherichia coli K12 in model food systems like milkand meat in vitro at temperatures as low as 158C (Walsh et al.,2008), and during sausage and cheese fermentation (Coc-concelli et al., 2003).

The use of natural antimicrobial molecules for inactivatingpathogenic microorganisms has received renewed attentiondue to concerns for toxicity of synthetic chemicals (Clevelandet al., 2001; Salamci et al., 2007). Plants have served as a sourcefor development of novel drugs, thereby contributing to humanhealth and well-being. A variety of plant-derived polyphenolsserve as dietary constituents as well as active components in anumber of herbal and traditional medicines. The antimicrobialproperties of several plant-derived essential oils have beendemonstrated (Burt, 2004; Holley and Patel, 2005) and a varietyof active components of these oils have been identified. Trans-cinnamaldehyde (TC) is an aromatic aldehyde present as amajor component of bark extract of cinnamon (Cinnamomumzeylandicum). Carvacrol (CAR) and thymol (THY) are antimi-crobial ingredients in oregano oil obtained from Origanumglandulosum. Similarly, eugenol (EUG) is an active ingredient inthe oil from cloves (Eugenia caryophillis). b-Resorcylic acid (BR;2, 4 dihydroxy benzoic acid) is a phytophenolic compoundwidely distributed among the angiosperms, and is a secondarymetabolite that plays a key role in the biochemistry and phys-iology of plants (Friedman et al., 2003). All the aforementionedmolecules are classified as generally regarded as safe by theU.S. Food and Drug Administration.

Previous research conducted in our laboratory revealedthat TC, EUG, CAR, and THY were effective in inactivatingmajor mastitis pathogens in milk (Ananda Baskaran et al.,2009), and TC inactivated Salmonella Enteritidis and Campy-lobacter jejuni in chicken drinking water (Kollanoor Johny et al.,2008). These compounds have also been reported to exhibitantimicrobial activity against Salmonella Typhimurium andSalmonella Typhimurium DT104 (Friedman et al., 2002; Si et al.,2006; Feng et al., 2007).

The objective of our study was to determine the efficacy ofsubinhibitory concentrations (SICs) of TC, EUG, THY, CAR,and BR independently or in combination in increasing thesensitivity of Salmonella Typhimurium DT104 to ampicillin,chloramphenicol, streptomycin, sulfonamide (sulfamethox-azole), and tetracycline.

Materials and Methods

Bacterial cultures and growth conditions

Two strains of Salmonella Typhimurium DT104, namely,H3380 (CDC, human strain) and 43 (pork strain), were used inthe study. Each strain was cultured separately in 10 mL oftryptic soy broth (TSB; Difco, Sparks, MD) at 378C for 24 hourswith agitation (100 rpm) to reach an optical density at 600 nm(OD600) of �0.7 yielding *9 log10 CFU=mL. The cultureswere sedimented by centrifugation (3600 g, 15 minutes, 48C),and the pellet was washed twice, resuspended in sterilephosphate-buffered saline (pH 7.3), and used as the inoculum.The bacterial population of the cultures was determined byplating 0.1 mL portions of appropriate dilutions on xyloselysine desoxycholate (XLD) agar (Difco) and tryptic soy agar(TSA) plates (Difco), with incubation at 378C for 24 hours.

Antibiotics and antibiotic resistance testing

Both strains of Salmonella Typhimurium DT104 were con-firmed for multiple antibiotic resistance by broth dilutionassay in 24-well tissue culture plates (CLSI, 2006). Briefly,duplicate wells containing 2 mL of TSB were inoculated with*6.0 log CFU of bacteria in the presence of antibiotics at theirrespective breakpoints for resistance, as described by CLSI(Rajic et al., 2004; CLSI, 2006; Farzan et al., 2008). Resistancebreakpoint of an antibiotic is defined as the concentration at orabove which the strain is resistant (Turnidge and Bell, 2005;Van Eldere, 2005). The antibiotics screened were ampicillin(Fisher Scientific, Fairlawn, NJ) at 32 mg=mL, chloramphenicol(Sigma Aldrich, St. Louis, MO) at 32 mg=mL, streptomycin(Fisher Scientific) at 32 mg=mL, sulfamethoxazole (ResearchProducts International Corp., Mt. Prospect, IL) at 512 mg=mL,and tetracycline (MP Biomedical Inc., Aurora, OH) at16 mg=mL. The cultures were incubated at 378C for 24 hours ina reciprocal shaker incubator (New Brunswick Scientific Co.Inc., Edison, NJ), followed by determination of OD600 in amicroplate reader (Model 550; Bio-Rad, Hercules, CA) (Nazeret al., 2005). The culture was also streaked on TSA and XLDagar plates supplemented with respective concentrations ofantibiotics, and incubated at 378C for 24 hours.

Plant molecules and determination of SICs

TC, CAR, THY, EUG, and BR were purchased from SigmaAldrich. Stock solutions of these molecules were prepared inabsolute ethanol (Nazer et al., 2005). To duplicate tissue cul-ture plate wells containing 2 mL of TSB inoculated with *6.0log CFU Salmonella Typhimurium DT104, 1–10 mL of stocksolutions of each molecule with an increment of 0.5 mL wasadded. The plates were then incubated in a shaker incubatorat 378C for 24 hours, and bacterial growth was monitored bydetermining OD600, and plating on TSA and XLD plates. Thehighest concentration of each plant molecule that did not in-hibit bacterial growth after 24 hours of incubation was se-lected as the SIC of the molecule. Duplicate samples wereincluded and the experiment was replicated three times.

Effect of plant molecules on antibiotic resistance

To determine if the plant molecules increased the sensi-tivity of Salmonella Typhimurium DT104 to antibiotics, the SICof each molecule was added to duplicate wells of 24-wellpolystyrene tissue culture plates containing 2 mL TSB inocu-lated with 0.1 mL (*6 log10 CFU) of the bacterial culture andsupplemented with each antibiotic at the respective break-point for resistance. Additionally, to study the synergisticeffect of the five molecules in combination (COMB) to increasethe sensitivity of Salmonella Typhimurium DT104 to ACSSuT,a cocktail containing each of the five compounds at less thanthe respective SIC was added to the wells. COMB containedTC, EUG, CAR, TH, and BR at final concentrations of85 mg=mL EUG, 84 mg=mL TC, 78 mg=mL CAR, 14mg=mL BR,and 12mg=mL THY in each treatment well. These concentra-tions were selected from a series of preliminary experiments,where several 10-fold diluted combinations of the five plantmolecules were tested for increasing the sensitivity of Salmo-nella Typhimurium DT104 to all the five antibiotics. Finalconcentration of alcohol in the treatment wells was 0.35%v=v.Suitable controls, including positive control (TSBþDT104),

1166 KOLLANOOR JOHNY ET AL.

negative control (TSB�DT104), positive antibiotic control(TSBþDT104, þantibiotic), negative antibiotic control (TSB�DT104, þantibiotic), positive molecule control (TSBþDT104,þmolecule), negative molecule control (TSB�DT104,þmolecule), and alcohol control (TSBþDT104, þ0.35% alco-hol), were included.

The tissue culture plates were incubated at 378C for 24 hoursin a reciprocal shaker incubator. Each well was mixed with a

1 mL pipette, and 0.2 mL portions were transferred to a 96-wellpolystyrene cell culture plate for OD600 determination in amicroplate reader (Nazer et al., 2005). Appropriate blanks wereincluded. Duplicate samples were included for each treatmentand the experiment was replicated three times.

Statistical analysis

For the absorbance data, each well was considered as anexperimental unit and a completely randomized 5�6 (fiveantibiotics and six treatments) factorial design was used. Thedata from three trials were averaged and analyzed with theProc-mixed version of Statistical Analysis Software (version9.2; SAS Institute Inc., Cary, NC). Differences among themeans were considered significant at p� 0.05 and were de-tected using Fisher’s least significant difference test, withappropriate corrections for multiple comparisons.

Results

The SICs of various plant molecules that allowed thegrowth of Salmonella Typhimurium DT104 isolates as in pos-itive control (TSBþDT104) are provided in Table 1. The

Table 1. Subinhibitory Concentrations of Plant

Molecules and Bacterial Counts

Counts (log10 CFU=mL)

Molecules SIC (mg=mL) XLD TSA

Trans-cinnamaldehyde 197 8.9� 0.02 8.9� 0.03Carvacrol 98 9.1� 0.04 9.0� 0.03Thymol 75 9.4� 0.02 9.5� 0.04Eugenol 133 8.7� 0.05 8.8� 0.06b-Resorcylic acid 90 9.2� 0.15 9.4� 0.10Control 9.0� 0.02 9.2� 0.00

SIC, subinhibitory concentration; XLD, xylose lysine desoxycho-late; TSA, tryptic soy agar.

FIG. 1. Effect of (A) trans-cinnamaldehyde (TC), (B) thymol (THY), (C) carvacrol (CAR), (D) b-resorcylic acid (BR), (E) eugenol(EUG), and (F) combination of five molecules (COMB) on increasing sensitivity of Salmonella Typhimurium DT104 strain H3380to antibiotics, ampicillin (Amp), chloramphenicol (Chl), streptomycin (Str), sulfamethoxazole (Sul), and tetracycline (Tet).a,b,cTreatment bars having different superscripts within a group of three, representing DT104þmolecule (molecule control[ctrl]), DT104þantibiotic (antibiotic control), and DT104þantibioticþmolecule (treatment), differ significantly at p< 0.05.

PLANT MOLECULES ENHANCE ANTIBIOTIC SENSITIVITY 1167

analysis of the absorbance data revealed a significant inter-action effect ( p< 0.0001) between the plant molecules andantibiotics in reducing the growth of the bacterium. AlthoughSalmonella Typhimurium DT104 grew in the presence of all theantibiotics, bacterial growth was significantly ( p< 0.05) lesserin the presence of tetracycline and chloramphenicol in com-parison to that with other antibiotics. However, in the pres-ence of ampicillin, streptomycin, or sulfamethoxazole,Salmonella Typhimurium DT104 grew to the maximum den-sity as in the positive controls. The mean absorbance in thenegative control (TSB alone) was zero at 0 and 24 hours ofincubation, and that in alcohol control (1.1� 0.02) was similarto the mean absorbance in positive control (1.1� 0.02).

The effect of various plant molecules and their combinationon the sensitivity of Salmonella Typhimurium DT104 strainH3380 to the five antibiotics is depicted in Figure 1A–F.Compared to the respective controls, TC increased the sensi-tivity of the pathogen to all the five antibiotics ( p< 0.05), asevidenced by the decreased absorbance values for samplescontaining the plant molecule and antibiotic (Fig. 1A). THYwas found to increase the sensitivity of the bacterium to all theantibiotics ( p< 0.05) except ampicillin (Fig. 1B), whereas CAR

made the pathogen sensitive to chloramphenicol, sulfa-methoxazole, and tetracycline ( p< 0.05) (Fig. 1C). BR was alsoeffective in increasing the sensitivity of the strain H3380 to allthe antibiotics ( p< 0.05), excluding tetracycline (Fig. 1D).However, EUG was least effective among the five plantmolecules, making the bacterium sensitive to only two anti-biotics ( p< 0.05), namely, chloramphenicol and sulfa-methoxazole (Fig. 1E). On the contrary, the COMB was veryeffective in modulating the antibiotic resistance of SalmonellaTyphimurium DT104 strain H3380; the pathogen growth wassignificantly suppressed ( p< 0.05) compared to the controls(Fig. 1F).

The effect of various plant molecules and their combinationon the sensitivity of Salmonella Typhimurium DT104 strain 43to the five antibiotics is depicted in Figure 2A–F. The effect ofTC and THY on increasing the sensitivity of strain 43 to an-tibiotics was similar to that observed with H3380. TC waseffective in increasing the sensitivity to all five antibiotics( p< 0.05) (Fig. 2A), whereas THY increased sensitivity to fourantibiotics ( p< 0.05) except ampicillin (Fig. 2B). EUG madethe strain more sensitive to chloramphenicol, streptomycin,and sulfamethoxazole ( p< 0.05) (Fig. 2E). Additionally, CAR

FIG. 2. Effect of (A) TC, (B) THY, (C) CAR, (D) BR, (E) EUG, and (F) COMB on increasing sensitivity of SalmonellaTyphimurium DT104 strain 43 to antibiotics Amp, Chl, Str, Sul, and Tet. a,b,cTreatment bars having different superscriptswithin a group of three, representing DT104þmolecule (molecule control), DT104þantibiotic (antibiotic control), andDT104þantibioticþmolecule (treatment), differ significantly at p< 0.05.

1168 KOLLANOOR JOHNY ET AL.

was significantly effective ( p< 0.05) in sensitizing the porkstrain to streptomycin and sulfamethoxazole (Fig. 2C) insteadof three antibiotics in H3380 (Fig. 1C). It was observed that BRcould increase the sensitivity of strain 43 to only sulfa-methoxazole ( p< 0.05) as against four antibiotics in strainH3380 (Fig. 2D). The COMB group significantly increased thesensitivity of the strain 43 to all antibiotics ( p< 0.05) com-pared to individual treatments.

Among the five antibiotics tested, ampicillin resistance wasfound to be the most difficult to modulate by the plant mol-ecules in both the strains of Salmonella Typhimurium DT104.However, TC and the combination of all the five plant mole-cules were able to reduce the ampicillin resistance of bothstrains significantly ( p< 0.05).

Discussion

A wide variety of antibiotics are used at subtherapeuticlevels in animal production worldwide (Sarmah et al., 2006;Silbergeld et al., 2008), favoring the selection of resistant bac-teria, including Salmonella Typhimurium DT104 in food ani-mals (Van Duijkeren et al., 2003; Varga et al., 2008). SinceSalmonella Typhimurium DT104’s resistance to multiple anti-biotics is chromosomally mediated, the removal of selectivepressure from these antibiotics would not possibly reverse theresistance (Humprey, 2001). Therefore, we investigated theefficacy of SICs of plant-derived antimicrobial molecules forincreasing the pathogen sensitivity to the five antibiotics, in-cluding ampicillin, chloramphenicol, streptomycin, sulfa-methoxazole, and tetracycline. Although numerous studieshave investigated the antimicrobial effect of a variety of plantmolecules on various pathogenic bacteria (Friedman et al.,2002, 2003; Si et al., 2006; Feng et al., 2007; Kollanoor Johnyet al., 2008; Ananda Baskaran et al., 2009), only limited infor-mation is available on their effect on bacterial antibiotic re-sistance. A recent study reported that TC decreasedclindamycin resistance in Clostridium difficile, a Gram-positivebacterium (Shahverdi et al., 2007).

Since the antibiotics and plant molecules were used at theirbreakpoints and SICs, respectively, the reduced growth ofSalmonella Typhimurium DT104 observed in the presence ofantibiotics and phytophenolics, in comparison to that insamples containing antibiotic or plant molecule alone, indi-cates increased pathogen sensitivity to the antibiotics medi-ated by the natural molecules. All the five testedphytophenolics were effective in making Salmonella Typhi-murium DT104 susceptible to one or more antibiotics. Amongthe various molecules, TC and THY were significantly( p< 0.05) more effective than the other phytophenolics inincreasing bacterial sensitivity to antibiotics. Moreover, thecombination of all the plant molecules at less than their re-spective SICs was highly effective ( p< 0.05) in reducing an-tibiotic resistance in the pathogen, thereby suggesting asynergistic effect among the phytophenolics.

Salmonella Typhimurium DT104 has been reported to pos-sess multiple genes encoding antibiotic resistance. These in-clude blaPSE-1 that encodes class A b-lactamase PSE-1, and ant(300)-Ia (aadA), encoding ANT (300)-I responsible for resistanceto streptomycin and spectinomycin by modifying differentresidues on the active sites of these antibiotics (Ridley andThrelfall, 1998; Briggs and Fratamico, 1999). In addition,DT104 expresses a cmlA homolog that mediates chloram-

phenicol resistance, sul1 that code for a dihydropteroatesynthase giving resistance to sulfonamides, and tetA that en-codes an efflux pump that mediates resistance to tetracycline(Briggs and Fratamico, 1999). Additional mechanisms thatconfer antibiotic resistance in bacteria include antibiotic-impermeable cell membrane or lack of transport systems nec-essary for antibiotic uptake. Moreover, bacteria could synthesizeinactivators=enzymes such as trans-peptidase (for ampicillinresistance), trans-acetylase (for chloramphenicol resistance), anda membrane-bound protein (for streptomycin resistance), ca-pable of deactivating antibiotics (Brooks et al., 1998).

Although it is not clear how the plant molecules decreasedthe antibiotic resistance of Salmonella Typhimurium DT104,the following potential mechanisms are suggested. Sinceplant essential oils and their components are hydrophobic innature, they primarily target the lipid-containing bacterialplasma membrane, making the membrane more permeable(Carson et al., 2002; Ultee et al., 2002). This change in perme-ability of bacterial plasma membrane brought about by theplant molecules could have permitted an increased uptake ofantibiotics by the bacterial cell. It is also possible that thephytophenolics at their SICs could have sublethally injuredSalmonella Typhimurium DT104, thereby increasing bacterialsensitivity to the antibiotics. Another plausible explanation isthat after entering the bacterial cell, the plant molecules in-hibited the efflux pumps responsible for reduced antibioticconcentration within the cell. Currently, further studies ex-ploring the mechanism(s) behind the phytophenolic-medi-ated antibiotic sensitivity in Salmonella Typhimurium DT104are underway in our laboratory.

Conclusions

Results of this study indicate that TC, THY, CAR, EUG, andBR were effective in enhancing Salmonella TyphimuriumDT104’s sensitivity to one or more antibiotics. These mole-cules could potentially be used as feed supplements to reducethe antibiotic resistance in Salmonella Typhimurium DT104 infood animals.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Kumar Venkitanarayanan, M.V.Sc., M.S., Ph.D.

Department of Animal ScienceUniversity of Connecticut

3636 Horsebarn Hill Road Ext., Unit 4040Storrs, CT 06269

E-mail: kumar.venkitanarayanan@uconn.edu

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