FOODBORNE PATHOGENS AND DISEASE Volume 11, Number 4, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/fpd.2013.1635

Influence of the Treatment of Listeria monocytogenes and Salmonella enterica Serovar Typhimurium with Citral on the Efficacy of Various Antibiotics Surama F. Zanini,1 Angela B. Silva-Angulo,2 Amauri Rosenthal,3 Dolores Rodrigo Aliaga,4 and Antonio Martı´nez 4

Abstract

The main goal of this work was to study the bacterial adaptive responses to antibiotics induced by sublethal concentration of citral on first-and second-generation cells of Listeria monocytogenes serovar 4b (CECT 4032) and Salmonella enterica serovar Typhimurium (CECT 443). The first-generation cells were not pretreated with citral, while the second-generation cells were obtained from cells previously exposed to citral during 5 h. The trials were conducted at 37C. The presence of citral in the culture medium and the antibiotic strips resulted in a reduced minimum inhibitory concentration (MIC) for the first-generation cells of Listeria monocytogenes serovar 4b and Salmonella Typhimurium. This result was observed for almost all the antibiotics, compared with the same microorganisms of the control group (without citral), which could represent an additive effect. For Listeria serovar 4b, the second-generation cells of the test group maintained the same susceptibility to antibiotics compared with cells in the control group and in the test group of the first generation. The second-generation cells of the control group indicated that the Salmonella Typhimurium maintained the same sensitivity to the antibiotics tested compared with the first generation of this group, except in the case of erythromycin, which exhibited an increased MIC value. With respect to the second-generation cells of Salmonella Typhimurium, the presence of citral determined a decrease in the antibiotic susceptibility for almost all of the antibiotics, except colistin, compared with the first-generation of the test group, which can be seen by increase of MIC values. In conclusion, the presence of citral in the culture medium of Listeria 4b and Salmonella Typhimurium increased the antibiotic susceptibility of the first generations, while we observed an increase in antibiotic resistance in the second generation of Salmonella Typhimurium. Introduction

M

embrane permeability barriers are among the factors contributing to the intrinsic resistance of bacteria to antibiotics. Some research has been conducted in recent years on the ability of natural antimicrobials derived from plants to increase the antibiotic efficacy of drug-resistant bacteria. Palaniappan and Holley (2010) reported a synergistic action among four natural antimicrobials and as many as three antibiotics to which tested bacteria were normally resistant. Moreover, several different types of plant-derived antibacterial compounds are used to ensure food quality and as preservatives for food safety. The concentrations of these compounds used in food products are mainly governed by their effect on the organoleptic properties. The concentrations needed to inhibit bacteria often exceed the flavor threshold that is ac-

ceptable to consumers. In this context, the antibacterial compounds are used at sublethal concentrations and in combination with nonthermal technologies (hurdle concept) to increase food safety (Pina-Pe´rez et al., 2009), or they are combined with major components of essential oils (EOs) to decrease their individual dosage, while producing the desired antibacterial effect, in a concentration that does not produce undesirable changes in the food flavor. Klein et al. (2013) verified that the EO compound combinations tested showed a higher antimicrobial effect than at single use. Despite these benefits, the exposure of bacteria to subinhibitory concentrations of antimicrobial compounds may activate intrinsic resistance mechanisms, thereby decreasing the susceptibility of the microbe to the inducing agent and concomitantly decreasing susceptibility to other unrelated antimicrobials. Mechanisms exist whereby microorganisms

1

Department of Veterinary Medicine, Espirito Santo Federal University, Alegre, Brazil. Departamento de Biotecnologı´a Agroalimentaria, Biopolis Sociedad Limitada, Paterna, Spain. 3 Embrapa Food Technology, Rio de Janeiro, Brazil. 4 Department of Preservation and Food Quality, Instituto de Agroquı´mica y Tecnologı´a de Alimentos–Consejo Superior de Investigaciones Cientificas (IATA-CSIC), Paterna, Spain. 2

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that are resistant to one antimicrobial may become resistant to others (Yousef and Juneja, 2003). It has been well established that bacterial cells can adapt and develop resistance to the bactericidal activity of physical or chemical treatments (Dubois-Brissonnet et al., 2011). Little is known about bacterial adaptation to plant-derived antimicrobials, notably in the case of foodborne pathogens, although some studies in the scientific literature have tried to clarify the situation. Escherichia coli strains that are resistant to thymol and eugenol were found to be more resistant to chloramphenicol (Walsh et al., 2003). It was observed by DuboisBrissonnet et al. (2011) that the tolerance to disinfectants of cells cultivated with sublethal concentrations of terpenes increased significantly for eugenol, thymol, carvacrol, and citral, probably due to alteration of the fatty acid composition. Alonso-Hernando et al. (2009) evaluated the influence of subinhibitory concentrations of different cleaners and disinfectants on the antibiotic sensitivity of four bacterial strains. The authors reported an increased resistance to streptomycin, chloramphenicol, and cephalothin in Listeria monocytogenes strains upon exposure to chemicals, especially hydrochloric acid. However, this type of selection, which has been demonstrated on numerous occasions, is not universal (Cottell et al., 2009). According to Rensch et al. (2013), only 25 strains of Salmonella enterica had the highest detected minimum inhibitory concentration (MIC) of 0.5 lg/mL triclosan, but an association with multidrug resistance could not be confirmed. The main goal of this work was to study the bacterial adaptive responses to antibiotics induced by sublethal concentration of citral on first- and second-generation cells of Listeria monocytogenes serovar 4b (CECT 4032) and Salmonella enterica serovar Typhimurium (CECT 443).

ZANINI ET AL.

(Somolinos et al., 2010). For this, citral was added to 20 mL of tryptone soya broth (TSB) in sterile flasks to obtain the indicated concentration (0.250 lL/mL). An aliquot of 5 mL of the overnight culture was added to each sample to give a final concentration of approximately 108 CFU/mL. Samples with citral and microorganism test were incubated at 37C. After citral treatment, there were two cell populations that resulted: death cells by citral action and the viable cells (damaged and nondamaged). Viable cells were recovered in TSB broth supplemented with 0.6% of yeast extract (TSB-YE) as a repair medium (Somolinos et al., 2010), following a protocol described by Chambliss et al. (2006). For that, the test cultures were pipetted into 50-mL centrifuge tubes and centrifuged for 15 min at 2795 · g at 4C in a Sorvall RC-513 Refrigerated Super-speed Centrifuge (Dupont Instruments, Newtown, CT). Following centrifugation, the supernatants were discarded, and the pellets were each suspended in 20 mL of TSB-YE. This process was repeated twice. The pellets were resuspended in the TSB-YE tubes and incubated at 37C for 12 h to obtain a second generation of cells. After this incubation period, the cell concentration was 8–8.5 log10 CFU/mL. First- and second-generation cell treatments

Materials and Methods

The first generation of cells was not pretreated with citral, while the second generation cells was obtained from cells previously exposed to citral during 5 h and recovered in TSBYE as a repair medium. Bacterial subcultures to be tested were made from the stock bacteria grown on TSB (Scharlab Chemie S.A.) by incubation at 37C for 12–14 h to produce a suspension of 8 log10 CFU/mL (first-generation cells) and from second generation cells with the 8–8.5 log10 CFU/mL concentration. The cell concentrations were confirmed by viable plate count (Saucedo-Reyes et al., 2009).

Chemicals

Experimental design

Citral was purchased as a mixture of the cis- and transisomers of 3,7-dimethyl-2,6-octadienal (95%) from Sigma Aldrich Company Ltd., Steinheim, Westphalia, Germany.

Test group. A total of 100 lL of an inoculum size of approximately 108 CFU/mL was grown on tryptone soya agar (TSA; Scharlab Chemie S.A.) supplemented with 0.6% of yeast extract (TSA-YE) in the presence of citral in the agar medium. The essential oil component was incorporated into the agar medium by using dimethyl sulfoxide (DMSO), following the method described by Firouzi et al. (1998). DMSO is frequently used as an oil solubilizer for natural measurement of the activity of an antimicrobial agent (Hili et al., 1997). In a preliminary study, no effect of antibacterial activity by DMSO alone was observed. The same result was previously verified by Rukayadi et al. (2009). The concentration of citral (0.250 lL/mL) was chosen on the basis of previous studies of sublethal damage and growth kinetics (Silva-Angulo et al., 2012) and was 10-fold lower than the MIC for citral described by Muriel-Galet et al. (2011) for L. monocytogenes and S. enterica.

Bacterial species and growth conditions of first-generation cells

Listeria serovar 4b (CECT 4032), associated with a case of meningitis after eating soft cheese, and Salmonella Typhimurium (CECT 443), related to a case of human food poisoning, were obtained from a pure lyophilized culture supplied by the Spanish Type Culture Collection. Glycerinated stocks of Listeria 4b (CECT 4032) and Salmonella Typhimurium (CECT 443) were generated. The microorganisms were obtained following the method described by Saucedo-Reyes et al. (2009). For this work, stock cultures at a concentration of approximately 8.5 · 108 colony-forming units (CFU/mL) were maintained in cryovials at - 80C. For both cell types, the average cell concentration of the vials was established by viable plate count, employing buffered peptone water (Scharlab Chemie S.A., Barcelona, Spain) for sample dilution. Bacterial species and growth conditions of second-generation cells

Bacterial overnight cultures at a concentration of 8 log10 CFU per mL were exposed to 0.250 lL/mL of citral for 5 h

Control group. A total of 100 lL of an inoculum size of 108 CFU/mL was grown on TSA-YE without citral. Antibiotic resistance evaluation and MIC determination

The MICs for erythromycin (macrolide), cephalothin (cephalosporin), gentamicin (aminoglycoside), bacitracin (polypeptide), colistin (lipopeptide), chloramphenicol (phenicol),

CITRAL AND ANTIBIOTICS INTERACTIONS

trimethoprim/sulfamethoxazole (folate pathway inhibitor), ampicillin (penicillin), and ciprofloxacin (fluoroquinolone) against the first- and second-generation cells of L. monocytogenes serovar 4b and Salmonella Typhimurium grown in TSA either without citral or with 0.250 lL/mL of citral were evaluated using the E-test method, in accordance with the manufacturer’s recommendations (bioMe´rieux). Inoculated Petri plates with and without citral and the antibiotic strips were incubated upside down at 37C for 48 h. The MIC values were read directly from the test strip, according to the manufacturer’s instructions, where the elliptical zone of inhibition intersected with the MIC scale on the strip. The MIC values were defined as the lowest concentration of an antimicrobial agent that prevented visible growth of the microorganism in an agar susceptibility test (CLSI, 2010). Statistical analysis

All experiments were repeated five times. Values are mean – standard deviations calculated by using the statistical software package Statgraphics Centurion XV (StatPoint Technologies, Inc., Warrenton, VA). Results

Tables 1 and 2 show the MIC for both generations of the Listeria 4b (CECT 4032) and Salmonella Typhimurium (CECT 443). According to the MIC interpretive criteria, our findings indicated that the first-generation of Listeria 4b was susceptible to erythromycin, gentamicin, chloramphenicol, ampicillin, and ciprofloxacin (Table 1). Erythromycin, gentamicin, and ampicillin had the lowest MIC value. Table 1 shows resistance to bacitracin and colistin, with MIC values > 8 lg/mL, and an intermediate response to trimethoprim/ sulfamethoxazole. For the first-generation of Listeria 4b, the presence of citral determined a reduction of the MIC for almost all of the antibiotics, except chloramphenicol, in comparison with the control group of this generation (Table 1). We also found very interesting results for colistin and bacitracin. Listeria 4b showed resistance to both antibiotics in the absence of citral, as observed in Table 1, but this behavior was reversed in the presence of citral, according to the MIC interpretive criteria used in this study. For Listeria 4b, the second-generation cells of the test group maintained the same susceptibility to antibiotics compared with cells in the control group and in the test group of the first-generation cells (Table 1). The presence of citral determined an increase of the antibiotic susceptibility for the second-generation of Listeria 4b, similar to that achieved in the first-generation cells, maintaining the reduction of MIC observed in comparison with the control group for all of the antibiotics except for ampicillin, for which the reduction was slightly lower than that observed for the first-generation cells (Table 1). For Salmonella, CLSI breakpoint patterns were used for all of the antimicrobials tested except erythromycin, bacitracin, and colistin; EUCAST (2013) breakpoints were used when CLSI breakpoints were not available for Salmonella (Table 2). Results indicated that the first generation of Salmonella Typhimurium was susceptible to ciprofloxacin, ampicillin, trimethoprim/sulfamethoxazole, gentamicin, chlorampheni-

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col, cephalothin, and colistin, with MIC values < 1 lg/mL. On the other hand, we observed resistances to erythromycin and bacitracin, with MIC values > 8 lg/mL (Table 2). The bacitracin and erythromycin resistances of Salmonella Typhimurium were not reversed in the presence of citral (Table 2), although a reduction of erythromycin MIC was observed, which still remains above the resistance breakpoint. For the first-generation of Salmonella Typhimurium, the presence of citral resulted in reduced MIC for almost all of the antibiotics, except for gentamicin, bacitracin, and colistin, which did not exhibit altered MIC compared with the control group. The second-generation cells of the control group indicated that the Salmonella Typhimurium maintained the same sensitivity to the antibiotics tested compared with the first generation of this group, except in the case of erythromycin, which exhibited an increased MIC value (Table 2). With respect to the second-generation cells of Salmonella Typhimurium, the presence of citral determined a decrease in the antibiotic susceptibility for almost all of the antibiotics, except colistin, as compared with the first-generation cells of the test group, shown as an increase of MIC values. Table 3 shows the fractional inhibitory concentration (FIC) for the various antibiotics tested. With respect to Listeria monocytogenes, increases in FIC were observed only for ampicillin and cephalothin, while in the case of Salmonella Typhimurium, increases in FIC were observed for all of the antibiotics tested except colistin and bacitracin. Discussion

Listeria 4b is closely associated with outbreaks and seems to possess greater virulence properties than the other serovars and is more related to food products ( Jay, 2005). Several serotypes of Salmonella enterica subspecies enterica can be transmitted from poultry meat and produce salmonellosis in humans. Enteritidis and Typhimurium are the most common serotypes associated with foodborne illness worldwide (WHO-GSS, 2004). The MICs of antibiotics for bacteria are the target value for the statistical planning of monitoring antimicrobial resistance studies (Aarestrup, 2004). To date, the majority of studies have relied on an increase in MIC to define an increase in resistance (Silva-Angulo et al., 2012). This study shows that Listeria 4b was susceptible to the antibiotics commonly used in veterinary and human listeriosis treatment (Orndorff et al., 2006). On the other hand, our findings indicated that Listeria 4b is resistant to bacitracin and colistin. However, these resistances were reversed in the presence of citral. In particular, the increase in susceptibility to colistin by citral could be of great interest because Grampositive bacteria are intrinsically resistant to this antibiotic, but it could be used against L. monocytogenes in combination with citral. It should be noted that the citral concentration used in this study only produces a bacteriostatic effect, which means that it does not kill microorganisms or prevent their growth, as was shown in previously published work (SilvaAngulo et al., 2012). With respect to bacitracin, Jay (2005) reported that, although most of the isolates of L. monocytogenes were susceptible to antibiotics that are effective against Gram-positive bacteria, L. monocytogenes resistance to bacitracin has been also reported. Moreover, in the presence of citral, there was an increased antibiotic efficacy against Listeria 4b by reduction of the

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0.016–256 0.016–256 0.016–256 0.016–256 0.016–256 0.016–256 0.002–32 0.016–256 0.002–32

Concentration range (lg/mL) £ 0.5 £8 £4 £1 £1 £4 £ 0.5 £2 £1

Susceptibility 1–4 16 8 >1–8 >1–8 8 1–2 n/a5 2

Intermediate ‡8 ‡ 32 ‡ 16 >8 >8 ‡ 16 ‡4 n/a5 ‡4

Resistance

0.125 – 0.017a 2.0 – 0.288a 0.125 – 0.037a 32.0 – 4.618a 96.0 – 18.475a 4.0 – 0.577a 1.0 – 0.144a 0.125 – 0.017 1.0 – 0.144

(no citral)

Control group

Mean values followed by different letters in the same row differ significantly by Fisher’s least significant difference test ( p £ 0.05). 1 Results obtained from Listeria 4b (CECT 4032). 2 Interpretive criteria of the MIC for Staphylococcus spp., as published in the current edition of CLSI (2007) document M100-S17. 3 Interpretive criteria of the MIC from the European Committee on Antimicrobial Susceptibility Testing—EUCAST (2013). 4 Interpretive criteria of the MIC for L. monocytogenes, as published in the current edition of CLSI (2006a, b) document M45-A. 5 CLSI breakpoints have not been established. n/a, not available.

Erythromycin2 Cephalothin2 Gentamicin2 Bacitracin3 Colistin3 Chloramphenicol2 Trimethoprim/ sulfamethoxazole4 Ampicillin4 Ciprofloxacin2

Antibiotics

MIC (lg/mL) interpretive criteria

0.064 – 0.017b 0.380 – 0.075b 0.064 – 0.009b 4.0 – 0.577b 4.0 – 0.577b 4.0 – 0.577a 0.047 – 0.009b 0.023 – 0.005b 0.38 – 0.075b

(0.250 lL/mL of citral)

Test group

First generation

0.126 – 0.005a 2.16 – 0.288a 0.128 – 0.037a 33.3 – 4.618a 98.2 – 11.351a 4.6 – 0.577a 1.16 – 0.144a 0.126 – 0.017a 1.3 – 0.014a

(no citral)

0.069 – 0.009b 0.39 – 0.075b 0.066 – 0.009b 4.1 – 0.577b 4.2 – 0.577b 4.4 – 0.577a 0.049 – 0.008b 0.047 – 0.008b 0.40 – 0.075b

(0.250 lL/mL of citral)

Test group

Second generation Control group

MIC (lg/mL)1

Table 1. Breakpoint Used in the Susceptibility Testing of Listeria monocytogenes and the Range of Values of the Minimum Inhibitory Concentration (MIC) of Antibiotics for the First Generation and Second Generation of L. monocytogenes Serovar 4b (CECT 4032) in Complete Culture Medium (Tryptone Soya Agar Supplemented with 0.6% of Yeast Extract) Using the E-Test Method in the Presence or Absence of Citral

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0.016–256 0.016–256 0.016–256 0.016–256 0.016–256 0.016–256 0.002–32 0.016–256 0.002–32

Concentration range (lg/mL) £1 £8 £4 £1 £1 £8 £2 £8 £1

Susceptibility >1–8 16 8 >1–8 >1–8 16 n/a5 16 2

Intermediate >8 ‡ 32 ‡ 16 >8 >8 ‡ 32 ‡4 ‡ 32 ‡4

Resistance

24.0 – 4.618c 2.0 – 0.288b 0.25 – 0.034b >256.0 – 0.00a 1.0 – 0.144a 4.0 – 0.577b 0.064 – 0.009b 0.38 – 0.075b 0.012 – 0.003b

No citral

Control group

0.250 lL/mL of citral

Test group

12.0 – 2.309d 0.25 – 0.034c 0.25 – 0.010b >256.0 – 0.0a 1.0 – 0.003a 2.0 – 0.288c 0.047 – 0.008c 0.19 – 0.037c 0.002 – 0.001c

Mean values followed by different letters in the same row differ significantly by Fisher’s least significant difference test ( p £ 0.05). 1 Results obtained from Listeria monocytogenes serovar 4b (CECT 4032). 2 Interpretive criteria of the MIC for Staphylococcus spp., as published in the current edition of CLSI (2007) document M100-S17. 3 Interpretive criteria of the MIC from the European Committee on Antimicrobial Susceptibility Testing—EUCAST (2013). 4 Interpretive criteria of the MIC for L. monocytogenes, as published in the current edition of CLSI (2006a, b) document M45-A. 5 CLSI breakpoints have not been established. n/a, not available.

Erythromycin2 Cephalothin2 Gentamicin2 Bacitracin3 Colistin3 Chloramphenicol2 Trimethoprim/ sulfamethoxazole4 Ampicillin4 Ciprofloxacin2

Antibiotics

MIC (lg/mL) interpretive criteria

First generation

64.0 – 9.237b 2.0 – 0.547b 0.25 – 0.005b >256.0 – 0.00a 1.0 – 0.010a 4.0 – 0.218b 0.064 – 0.018b 0.38 – 0.075b 0.012 – 0.001b

No citral

96.0 – 10.152a 3.0 – 0.577a 0.38 – 0.075a >256.0 – 0.00a 1.0 – 0.041a 6.0 – 1.154a 0.25 – 0.034a 1.0 – 0.144a 0.023 – 0.006a

0.250 lL/mL of citral

Test group

Second generation Control group

MIC (lg/mL)1

Table 2. Breakpoints Used in the Susceptibility Testing of Salmonella spp. and the Range of Values of the Minimum Inhibitory Concentration (MIC) of Antibiotics for the First Generation and Second Generation of Salmonella enterica Serovar Typhimurium (CECT 443) in Complete Culture Medium (Tryptone Soya Agar Supplemented with 0.6% of Yeast Extract) Using the E-Test Method in the Presence or Absence of Citral

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Table 3. Fractional Inhibitory Concentration for Different Antibiotics Tested in Presence of Citral Listeria monocytogenes Antibiotics Erythromycin Cephalothin Gentamicin Bacitracin Colistin Chloramphenicol Trimethoprim/sulfamethoxazole Ampicillin Ciprofloxacin

Salmonella enterica

First generation

Second generation

First generation

Second generation

0.51 0.19 0.51 0.13 0.04 1.00 0.05 0.18 0.38

0.51 0.25 0.51 0.13 0.04 1.00 0.05 0.37 0.38

0.50 1.12 1.00 1.00 1.00 0.50 0.70 0.50 0.10

1.50 2.00 1.50 1.00 1.00 1.50 3.90 2.60 2.80

MIC for almost all the antibiotics, except chloramphenicol. Citral probably acts as an absorption enhancer for drugs because of its low toxicity and ability to increase the permeability of both hydrophilic and lipophilic molecules (ElKattan et al., 2000). The results obtained with control and test groups of the second-generation Listeria cells appear to indicate that the 5-h pretreatment with citral did not result in increased resistance to the antibiotics tested. Nevertheless, the result obtained for ampicillin deserves further study, considering more cell generations by continued use of this natural antimicrobial in relation to this antibiotic. Our findings show that Salmonella Typhimurium was sensitive to antibiotic therapy indicated in human salmonellosis (Ziprin and Hume, 2001). However, it was resistant to erythromycin and bacitracin. These resistances were an expected result because erythromycin and bacitracin have no action against Gram-negative bacteria. Salmonella has intrinsic mechanisms of resistance to erythromycin owing to its low permeability to antibiotics, which is a common property of Gram-negative bacteria (Fluit et al., 2001). In the first-generation cells of Salmonella Typhimurium, the sublethal concentration of citral becomes more susceptible to the antibiotics ciprofloxacin, ampicillin, trimethoprim/sulfamethoxazole, chloramphenicol, cephalothin, and erythromycin, although the MIC for this last antibiotic still remains above the resistance breakpoint and very close to the intermediate resistance. Moreover, the result obtained with the control group of the second-generation cells of Salmonella Typhimurium is important because it appears to indicate that 5 h of pretreatment with citral affects the response of the microorganisms to some antibiotics. It is especially important in the case of erythromycin because a cross-resistance could take place between the natural antimicrobial substance and the antibiotic. Physiological responses are implicated in the increase of bacterial survival and tolerance in harsh environments, including protein upregulation or downregulation, modifications to the cell-membrane composition, and altered morphology. In addition, these physiological changes following stressful conditions can induce cross-resistance to other stressful environmental conditions, modifications to colonization, or virulence (Dubois-Brissonnet et al., 2011). The extent to which bacteria can adapt to the presence of EOs is also important for further evaluation. For the secondgeneration cells of Salmonella Typhimurium, the presence of citral determined an increase in the MIC values for almost

all the antibiotics, except colistin, compared with the values for the first generation. This result is interesting because it indicates that 5 h of pretreatment with citral produces some changes in second-generation Salmonella Typhimurium that make the serovar less susceptible to some antibiotics. Therefore these data are useful in improving background data on risk assessment. Furthermore, these results could indicate an interrelationship between natural antimicrobials and antibiotics. The FIC relationship was obtained with the following formula: The MIC of the antibiotic in combination with the antimicrobial substance divided by the MIC of the antibiotic alone. In our results, it was verified that FIC values > 1 indicate an increase in resistance to the antibiotic, while values < 1 indicate a decrease in resistance. As can be seen, Salmonella Typhimurium reacted to sublethal antimicrobial treatments faster than Listeria monocytogenes. The results obtained are in accordance with other reports (DuboisBrissonnet et al., 2011), particularly the results shown in this article for Salmonella Typhimurium, suggesting that this microorganism could be slowly becoming antibiotic resistant, as demonstrated by the increase in MIC values for second-generation cells. Preservative factors can impose nonlethal stresses over bacteria in foods while potentially eliciting stress tolerance. Because these cumulative processes are all applied at subinhibitory concentrations, they may promulgate an adaptive stress response and enable the survival of a greater fraction of stressed bacterial population (Yousef and Juneja, 2003). Also of potential concern is the possibility that the widespread use of natural antimicrobials at sublethal concentrations could be responsible for the selection and maintenance of antibiotic-resistant bacteria. Conclusions

The presence of citral in the culture medium of Listeria 4b (CECT 4032) and Salmonella Typhimurium (CECT 443) increased antibiotic effectiveness against the first generation of cells; however, the second generation of Salmonella Typhimurium cells exhibited increased resistance after previous exposure to citral. These results clearly indicate that the effect of pretreatment on the susceptibility of the microorganisms to antibiotics and on the antimicrobial/antibiotic interaction could be microorganism dependent. Consequently, studies to evaluate the effect of sublethal concentrations of natural antimicrobials on the response of microorganisms to antibiotics should be conducted on a microorganism–antimicrobial– antibiotic basis.

CITRAL AND ANTIBIOTICS INTERACTIONS Acknowledgments

This work was supported by CNPq (a Postdoctoral Fellowship Program), Embrapa Labex Europa, and Intituto de Agroquı´mica y Technologı´a de Alimentos–Consejo Superior de Investigaciones Cientificas (IATA-CSIC), which provided S. F. Zanini with a grant to carry out this research, and by Project AGL 2010-22206-Co2-01 and the Fondo Estructural Europeo de Desarrollo Regional program. Disclosure Statement

No competing financial interests exist. References

Aarestrup FM. Monitoring of antimicrobial resistance among food animals: Principle and limitations. J Vet Med B 2004; 51:380–388. Alonso-Hernando A, Capita R, Prieto M, Alonso-Calleja C. Comparison of antibiotic resistance patterns in Listeria monocytogenes and Salmonella enterica strains pre-exposed and exposed to poultry decontaminants. Food Control 2009;20:1108–1111. Chambliss LS, Narang N, Juneja VK, Harrison MA. Thermal injury and recovery of Salmonella enterica serovar Enteritidis in ground chicken with temperature, pH, and sodium chloride as controlling factors. J Food Prot 2006;69:2058–2065. [CLSI] Clinical and Laboratory Standards Institute. Methods for Antimicrobial Dilution and Disk Susceptibility Testing of Infrequently Isolated or Fastidious Bacteria; Approved Guideline M45-A. Wayne, PA: CLSI, 2006a. CLSI. Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard M2-A9. Wayne, PA: CLSI, 2006b. CLSI. Performance Standards for Antimicrobial Susceptibility Testing: Seventeenth Informational Supplement M100-S17. Wayne, PA: CLSI, 2007. CLSI. Performance Standards for Antimicrobial Susceptibility Testing: Twentieth Informational Supplement M100-S20. Wayne, PA: CLSI, 2010. Cottell A, Denyer SP, Hanlon GW, Ochs D, Maillard J-Y. Triclosan-tolerant bacteria: Changes in susceptibility to antibiotics. J Hosp Infect 2009;72:71–76. Dubois-Brissonnet F, Naitali M, Mafu AA, Briandet R. Induction of fatty acid composition modifications and tolerance to biocides in Salmonella enterica serovar Typhimurium by plant-derived terpenes. Appl Environ Microbiol 2011;77: 906–910. El-Kattan AF, Asbill CS, Michniak BB. The effect of terpene enhancer lipophilicity on the percutaneous permeation of hydrocortisone formulated in HPMC gel systems. Int J Pharm 2000;198:179–189. [EUCAST] European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters, version 3.0, 2013. Available at: http://www.eucast .org/clinical_breakpoints, accessed January 1, 2013. Firouzi R, Azadbakht M, Nabinedjad A. Anti-listerial activity of essential oils of some plants. J Appl Anim Res 1998; 14:75–80. Fluit AC, Visser MR, Schmitz FJ. Molecular detection of antimicrobial resistance. Clin Microbiol Rev 2001;14:836–871. Hili P, Evans CS, Veness RG. Antimicrobial action of essential oils: The effect of dimethylsulphoxide on the activity of cinnamon oil. Lett Appl Microbiol 1997;24:269–275. Jay JM. Microbiologia de Alimentos [Food Microbiology], 6th ed. Porto Alegre, Brasil: Artmed, 2005.

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Klein G, Ruben C, Upmann M. Antimicrobial activity of essential oil components against potential food spoilage microorganisms. Curr Microbiol 2013;67:200–208. Muriel-Galet V, Cerisuelo JP, Lo´pez-Carballo G, Aucejo S, Gavara R, Herna´ndez-Mun˜oz P. Evaluation of EVOH-coated PP films with oregano essential oil and citral to improve the shelf-life of packaged salad. Food Control 2011;30:137–143. Orndorff P, Hamrick T, Smoak I, Havell E. Host and bacterial factors in listeriosis pathogenesis. Vet Microbiol 2006;114:1–15. Palaniappan K, Holley RA. Use of natural antimicrobials to increase antibiotic susceptibility of drug resistant bacteria. Int J Food Microbiol 2010;140:164–168. Pina-Pe´rez MC, Silva-Angulo AB, Muguerza-Marquı´nez B, Rodrigo Aliaga D, Martı´nez Lo´pez A. Synergistic effect of high hydrostatic pressure and natural antimicrobials on inactivation kinetics of Bacillus cereus in a liquid whole egg and skim milk mixed beverage. Foodborne Pathog Dis 2009;6:649–656. Rensch U, Klein G, Schwarz S, Kaspar H, De Jong A, Kehrenberg C. Comparative analysis of the susceptibility to triclosan and three other biocides of avian Salmonella enterica isolates collected 1979 through 1994 and 2004 through 2010. J Food Prot 2013;76:653–656. Rukayadi Y, Lee K, Han S, Yong D, Hwang JK. In vitro activities of panduratin A against clinical Staphylococcus strains. Antimicrob Agents Chemother 2009;53:4529–4532. Saucedo-Reyes D, Marco-Celdra´n A, Pina-Pe´rez MC, Rodrigo D, Martı´nez Lo´pez A. Modeling survival of high hydrostatic pressure treated stationary and exponential phase Listeria innocua cells. Innovative Food Sci Emerging Tech 2009;10:135–141. Silva-Angulo A, Belda-Galbis CM, Zanini SF, Rodrigo D, Martorell P, Martı´nez A. Sublethal damage in Listeria monocytogenes after non-thermal treatments, and implications for food safety. In: Listeria Infections: Epidemiology, Pathogenesis and Treatment. Romano A, Giordano CF (eds.). Hauppauge, NY: Nova Science Publishers, 2012. Somolinos M, Garcia D, Condon S, Mackey BM, Pagan R. Inactivation of Escherichia coli by citral. J Appl Microbiol 2010;108:1928–1939. Walsh SE, Maillard J-Y, Russell AD, Charbonneau DL, Bartolo RG, Catrenich C. Development of bacterial resistance to several biocides and effects on antibiotic susceptibility. J Hosp Infect 2003;55:98–107. [WHO-GSS] World Health Organization–Global Salm-Surv. Top 15 Salmonella serotype list. Available at: http://sherlock .dzc.dk/pls/portal30/ARJ.ALL_SERO_SUMMARIES_REP .SHOW_PARMS, accessed September 10, 2004. Yousef A, Juneja VK. Microbial Stress Adaptation and Food Safety. Boca Raton, FL: CRC Press, 2003. Ziprin RL, Hume MH. Human salmonellosis: General medical aspects. In: Foodborne Disease Handbook: Bacterial Pathogens. Hui YH, Pierson MD, Gorham RJ (eds.). New York: Marcel Dekker, Inc., 2001, pp. 285–321.

Address correspondence to: Antonio Martı´nez, PhD Department of Preservation and Food Quality Instituto de Agroquı´mica y Tecnologı´a de Alimentos–Consejo Superior de Investigaciones Cientificas (IATA-CSIC) Avenida Agustı´n Escardino, 7 46980 Paterna, Spain E-mail: [email protected]

Influence of the treatment of Listeria monocytogenes and Salmonella enterica serovar Typhimurium with citral on the efficacy of various antibiotics.

The main goal of this work was to study the bacterial adaptive responses to antibiotics induced by sublethal concentration of citral on first-and seco...
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