Essential oil production in shoot cultures versus field-grown plants of Thymus caespititius

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6–12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC–mass spectrometry (GC–MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17–25 % in the case of carvacrol and 18–23 % in that of thymol, closely followed by carvacryl acetate (15–23 %) and thymyl acetate (11–15 %). For the SC chemotype, carvacrol (13–28 %), sabinene (18–45 %), and thymol (9–12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences.     

Rapid Identification and Typing of Listeria Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Listeria monocytogenes is a food-borne pathogen that is the causative agent of human listeriosis, an opportunistic infection that primarily infects pregnant women and immunologically compromised individuals. Rapid, accurate discrimination between Listeria strains is essential for appropriate therapeutic management and timely intervention for infection control. A rapid method involving matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) that shows promise for identification of Listeria species and typing and even allows for differentiation at the level of clonal lineages among pathogenic strains of L. monocytogenes is presented. A total of 146 strains of different Listeria species and serotypes as well as clinical isolates were analyzed. The method was compared with the pulsed-field gel electrophoresis analysis of 48 Listeria strains comprising L. monocytogenes strains isolated from food-borne epidemics and sporadic cases, isolates representing different serotypes, and a number of Listeria strains whose genomes have been completely sequenced. Following a short inactivation/extraction procedure, cell material from a bacterial colony was deposited on a sample target, dried, overlaid with a matrix necessary for the MALDI process, and analyzed by MALDI-TOF MS. This technique examines the chemistry of major proteins, yielding profile spectra consisting of a series of peaks, a characteristic “fingerprint” mainly derived from ribosomal proteins. Specimens can be prepared in a few minutes from plate or liquid cultures, and a spectrum can be obtained within 1 minute. Mass spectra derived from Listeria isolates showed characteristic peaks, conserved at both the species and lineage levels. MALDI-TOF MS fingerprinting may have potential for Listeria identification and subtyping and may improve infection control measures.     

Essential oil variability in Thymbra capitata (L.) Cav. growing wild in Southern Apulia (Italy)

Essential oils of Thymbra capitata (Thymus capitatus) collected from Southern Apulia (Italy) were analysed using gas chromatography and gas chromatography–mass spectrometry techniques, to check for chemical variability. The study showed that among the 75 components of the oils the most recurrent ones were thymol and carvacrol, which always constituted more than 50% of the oils, as well as γ-terpinene, borneol and p-cymene. Cluster analysis led to the identification of three chemotypes: thymol, carvacrol and thymol/carvacrol; this was presumably a crossbreed between the other two chemotypes. Principal component analysis showed the direct correlation among myrcene, α-terpinene and γ-terpinene; anti-correlation between thymol and carvacrol, and the inverse correlation between linalool and myrcene. Moreover, lower thymol concentrations were accompanied by an increase in myrcene, α-terpinene and γ-terpinene.     

Suppression of Listeria monocytogenes Scott A in Fluid Milk by Free and Liposome-Entrapped Nisin

Nisin is an antimicrobial polypeptide inhibitory toward Gram-positive bacterial pathogens, including Listeria monocytogenes. Encapsulating nisin in lipid nanocapsules (i.e., liposomes) has been shown to protect antimicrobial functionality in complex food matrices. The capacity of liposomes to encapsulate a fluorescent reporter was determined via spectroscopy. Survival and growth of L. monocytogenes incubated in fluid milk containing 50 IU/ml free or liposome-entrapped nisin was assayed via periodic enumeration of survivors. Liposomes were formulated from phosphatidylcholine (PC) and phosphatidyl-DL-glycerol (PG) and prepared as PC, PC/PG 7/3 or PC/PG 6/4 (mol. fraction). Antilisterial activity of nisin-loaded liposomes was determined in ultra-high temperature processed fluid milk containing approximately 4.0 log₁₀ CFU/ml L. monocytogenes Scott A plus liposomal or free nisin at 50 IU/mL. Samples were aerobically held at 5 or 20°C; L. monocytogenes were enumerated via plating after 0, 1, 3, 6, 12, 24, 48, and 72 incubation hours. Liposome entrapment did not enhance pathogen inhibition when compared to free nisin as a function of storage temperature or incubation duration.     

Comparison of the Potency of the Lipid II Targeting Antimicrobials Nisin, Lacticin 3147 and Vancomycin Against Gram-Positive Bacteria

While nisin (lantibiotic), lacticin 3147 (lantibiotic) and vancomycin (glycopeptides) are among the best studied lipid II-binding antimicrobials, their relative activities have never been compared. Nisin and lacticin 3147 have been employed/investigated primarily as food preservatives, although they do have potential in terms of veterinary and clinical applications. Vancomycin is used exclusively in clinical therapy. We reveal a higher potency for lacticin 3147 (MIC 0.95?C3.8???g/ml) and vancomycin (MIC 0.78?C1.56???g/ml) relative to that of nisin (MIC 6.28?C25.14???g/ml) against the food-borne pathogen Listeria monocytogenes. A comparison of the activity of the three antimicrobials against nisin resistance mutants of L. monocytogenes also reveals that their susceptibility to vancomycin and lacticin 3147 changed only slightly or not at all. A further assessment of relative activity against a selection of Bacillus cereus, Enterococcus and Staphylococcus aureus targets revealed that vancomycin MICs consistently ranged between 0.78 and 1.56???g/ml against all but one strain. Lacticin 3147 was found to be more effective than nisin against B. cereus (lacticin 3147 MIC 1.9?C3.8???g/ml; nisin MIC 4.1?C16.7???g/ml) and E. faecium and E. faecalis targets (lacticin 3147 MIC from 1.9 to 3.8???g/ml; nisin MIC ??8.3???g/ml). The greater effectiveness of lacticin 3147 is even more impressive when expressed as molar values. However, in agreement with the previous reports, nisin was the more effective of the two lantibiotics against S. aureus strains. This study highlights that in many instances the antimicrobial activity of these leading lantibiotics are comparable with that of vancomycin and emphasizes their particular value with respect to use in situations including foods and veterinary medicine, where the use of vancomycin is not permitted.     

Acaricidal potential of some essential oils and their monoterpenoids against the two-spotted spider mite Tetranychus urticae (Koch.)

Abstract

The acaricidal potential of fourteen essential oils and fourteen of their major monoterpenoids were tested against the Two-spotted spider mite Tetranychus urticae. Two different time intervals of 24 and 48 hrs were used in evaluation. The assays after 24 hrs showed that mattercary, fennel, caraway, garlic, cinnamon, chenopodium and eucalyptus were highly potent. As for monoterpenoids, chlorothymol was found to be the most effective against T. urticae followed by thymol, carvacrol and cinnamaldehyde.     

Development of a Novel Selective and Differential Medium for the Isolation of Listeria monocytogenes

A new medium (lecithin and levofloxacin [LL] medium) is described for the isolation of Listeria monocytogenes from food samples. LL medium includes lecithin from soybeans for the detection of phosphatidylinositol-specific phospholipase C (PI-PLC) and phosphatidylcholine-specific phospholipase C (PC-PLC) produced by L. monocytogenes. Levofloxacin is incorporated to inhibit the growth of microorganisms other than L. monocytogenes, especially Bacillus cereus, shown to possess PI-PLC and PC-PLC activities. L. monocyogenes produced white colonies with a halo on LL medium, whereas Listeria innocua appeared as white colonies without a halo. Levofloxacin at 0.20 mg/liter completely inhibited the growth of B. cereus, while the growth of L. monocytogenes was unaffected. In the second phase of the study, the sensitivity and the specificity of LL medium were compared to those of modified Oxford agar (MOX) and two chromogenic media (Brilliance Listeria agar and CHROMagar Listeria), using a total of 250 food samples. From 200 unspiked food samples, the specificity of LL medium (96.0%) was superior to that of MOX (72.0%) and similar to the specificities of Brilliance Listeria agar (96.5%) and CHROMagar Listeria (94.5%). From 50 spiked food samples, LL medium and CHROMagar Listeria represented the highest sensitivities (96.0%), followed by Brilliance Listeria agar (92.0%) and MOX (54.0%). Also, LL medium showed the highest confirmation rate (98.8%), followed by Brilliance Listeria agar (98.7%), CHROMagar Listeria (98.3%), and MOX (52.0%). On the basis of its good specificity and cost effectiveness, LL medium is useful for the isolation of L. monocytogenes from food samples.     

Influence of planktonic and sessile Listeria monocytogenes on Caenorhabditis elegans

Listeria monocytogenes is the etiologic agent of listeriosis, a food-borne disease affecting humans and a variety of animals. In order to combat this pathogen, it is crucial to have an understanding of its natural interplay with the environment. For this reason, the free soil nematode Caenorhabditis elegans was focused upon because of its shared natural habitat with Listeria and its potential as a model organism for Listeria pathogenesis. Previous studies have generated some contradictory results on Listeria’s ability to kill C. elegans, making additional interaction studies such as this more attractive. In our study, we carried out a series of killing assays in a systematic manner using different Listeria strains under different growth conditions. In addition to studying the effects of planktonic cells, we examined the interaction between C. elegans and sessile listerial cells. Our findings suggest that, rather than causing infection and death, L. monocytogenes may extend the life span of C. elegans. This indicates that Listeria is not pathogenic to C. elegans. We also found that C. elegans can feed and ingest sessile cells, as well as carry the pathogen in its gut, implying that C. elegans could be a vehicle for L. monocytogenes spread in the environment.     

Nisin A and Polymyxin B as Synergistic Inhibitors of Gram-positive and Gram-negative Bacteria

Nisin A and polymyxin B were tested alone and in combination in order to test their antagonism against Listeria innocua HPB13 and Escherichia coli RR1, respectively. While the combination of both antibacterial substances was synergistically active against both target bacteria, nisin A alone did not show any inhibition of E. coli RR1. The nisin A/polymyxin B combination at 1.56/2.5 μg ml^?1 caused lysis of about 35.86 ± 0.35 and 73.36 ± 0.14% of L. innocua HPB13 cells after 3 and 18 h, respectively. Polymyxin B at 0.12 μg ml^?1 and nisin A/polymyxin B at 4.64/0.12 μg ml^?1 decreased the numbers of viable E. coli RR1 cells by about 0.23 and 0.65 log₁₀ CFU ml^?1, respectively, compared to the control. Our data suggest that the concentration of nisin A required for the effective control of pathogenic strains Listeria spp. could be lowered considerably by combination with polymyxin B. The use of lower concentrations of nisin A or polymyxin B should slow the emergence of bacterial populations resistant to these agents.     

Influence of environmental and genetic factors on the stability of essential oil composition of Thymus pulegioides

Thymus pulegioides plants were collected from various natural habitats of Lithuania and transferred into a new uniform environment. The plants were cloned annually at controlled conditions and their essential oil composition was monitored by capillary GC and GC/MS. The geraniol/geranial/neral (G/G/N), thymol (T), linalool (L), carvacrol/γ-terpinene/p-cymene (C/γT/pC) and thymol/carvacrol/γ-terpinene/p-cymene (T/C/γT/pC) chemotypes of T. pulegioides were studied. It was found that according to the stability of essential oil composition on a sudden change of environmental conditions two types of T. pulegioides can be distinguished: (1) plants which preserve their chemical composition of the essential oils; (2) plants, which considerably change their chemical composition of the essential oils.     

Glutamate Decarboxylase-Mediated Nisin Resistance in Listeria monocytogenes

Analysis of a complete set of glutamate decarboxylase (gad) mutants of Listeria monocytogenes strain LO28 (ΔgadD1, ΔgadDT1, ΔgadD2, ΔgadT2, and ΔgadD3 mutants) revealed that the ΔgadD1 mutant is impaired in its ability to tolerate exposure to both sublethal and lethal levels of the lantibiotic nisin. gadD1 is strain variable and is found only in approximately 50% of L. monocytogenes strains. Growth and survival experiments revealed that possession of gadD1 correlates with a higher degree of tolerance to nisin. Significantly, a similar finding using a gadB mutant of L. lactis IL1403 implies that this may be a general phenomenon in Gram-positive bacteria. Our findings thus suggest that the specific inhibition of GAD activity or a reduction in the levels of free glutamate may prevent the growth of otherwise resistant GAD⁺ bacteria in foods where low pH and/or nisin is used as a preservative.Listeria monocytogenes is a food-borne pathogen that is the causative agent of listeriosis, an opportunistic infection associated with high rates of morbidity and mortality (18). The microorganism has also been the cause of significant commercial losses, being responsible for 71% of all recalls of food products due to bacterial contamination in the United States between 1993 and 1998 (25). The ubiquitous nature of L. monocytogenes, together with its ability to tolerate a variety of environmental extremes, including high salt concentrations and low pH, and the ability to grow at refrigeration temperatures, makes control of the bacterium in foods difficult (10). Hence it is not altogether surprising that the food industry invests considerable effort into developing strategies to prevent the survival and growth of this pathogen. One such strategy involves the utilization of bacteriocins. Bacteriocins are antimicrobial peptides produced by one bacterium that inhibit the growth of other bacteria and have been used as “natural” preservatives to control undesirable microbiota in food (5). The most extensively studied bacteriocin is nisin A (here referred to as nisin), a 34-amino-acid class I bacteriocin (lantibiotic) produced by Lactococcus lactis strains that is currently approved for use in foods in over 50 countries. Nisin functions by binding lipid II, an essential precursor of cell wall peptidoglycan biosynthesis. Binding to lipid II also facilitates the formation of pores within the cytoplasmic membrane leading to the release of ATP and other small cytoplasmic contents, resulting in depolarization of the membrane potential and ultimately cell death (13).The molecular mechanisms employed by L. monocytogenes to cope with nisin are poorly understood. To date, loci that have been implicated in nisin tolerance include the alternative stress sigma factor SigB, the class three stress gene regulator CtsR, the two-component systems LisRK and HK1027, and a penicillin binding protein, Pbp (2, 6, 11, 15, 21). In addition, several studies have uncovered a link between the acid stress response of L. monocytogenes and nisin resistance (3, 17, 24). Several systems are employed by L. monocytogenes to withstand low pH stress, but the glutamate decarboxylase (GAD) system is probably the most important (an overview of the GAD system is in Fig. ​Fig.1).1). Mutation of specific gad genes renders cells exquisitely sensitive to ex vivo porcine and synthetic human gastric fluid and significantly impairs growth and survival in low-pH foods (4, 7, 8). Given the link between acid and nisin resistance phenotypes, the present study was initiated in order to investigate the contribution, if any, of gad genes to the nisin tolerance of L. monocytogenes.Open in a separate windowFIG. 1.An overview of the L. monocytogenes glutamate decarboxylase (GAD) system. L. monocytogenes possesses five gad genes. gadD1, gadD2, and gadD3 encode decarboxylases which catalyze the conversion of glutamate to γ-amino butyrate (GABA) and carbon dioxide (CO₂). gadT1 and gadT2 encode glutamate-GABA antiporters. Nisin functions by binding to lipid II, an essential precursor of cell wall peptidoglycan synthesis. Binding to lipid II facilitates the formation of pores within the cytoplasmic membrane leading to the release of ATP and may ultimately result in cell death. We suggest that under certain conditions gadD1 may contribute to intracellular ATP pools and hence tolerance of nisin.     

Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.     

Novel cost-efficient real-time PCR assays for detection and quantitation of Listeria monocytogenes

Listeriosis is a serious food-borne infection with mortality rates approaching 30%. Therefore, the rapid, cost-effective, and automated detection of Listeria monocytogenes throughout the food chain continues to be a major concern. Here we describe three novel quantitative real-time PCR assays for L. monocytogenes based on amplification of a target hlyA gene with SYBR Green I chemistry and hydrolysis probe (TaqMan MGB probe). In order to offer sensitive, rapid and robust tool of additional economical value the real-time PCR assays were designed and optimized to only 5 μl-reactions. All assays were evaluated by using different non-reference Listeria strains isolated from various food matrices. Results demonstrated specificity to L. monocytogenes with accurate quantification over a dynamic range of 5-6 log units with R² higher than 0.98 and amplification efficiencies reaching above 92%. The detection and quantification limits were as low as 165 genome equivalents. Comparison of novel assays to commercially available TaqMan® Listeria monocytogenes Detection Kit and previously published studies revealed similar specificity, sensitivity and efficiency, but greater robustness and especially cost-efficiency in the view of smaller reaction volumes and continuous increase in sample throughput.     

Comparative analysis of essential oil compositions in seven populations of Bakhtiarian savory in natural and field conditions

Bakhtiarian savory (Satureja bachtiarica Bunge) is an endemic herb growing wild in Iran with interesting pharmacological and biological properties. In this research, the variability of essential oil content and phytochemical compositions among seven populations of this species was studied in both natural and field conditions. There was a wide interpopulation variability in phytochemical variation of studied populations under both natural and field conditions, indicating the existence of different chemotypes. Six chemical compositions showed high value in studied plants so that carvacrol and thymol were known as major compounds and confirmed by principal component analysis. Ilam population had the highest thymol in both field (77.10 %) and natural (42.32 %) conditions and was known as a thymol chemotype. Beside, Yazd and Shahrekord populations, respectively, showed the highest carvacrol percentages in both field (84.83 and 63.81 %) and natural (72.50 and 58.05 %) conditions and were known as carvacrol chemotypes. In other populations, carvacrol and thymol were the main components and showed partly equal percentage and these populations were known as carvacrol/thymol chemotypes. According to Pearson correlation analysis, carvacrol was negatively correlated with p-cymene, γ-terpinene and thymol (r = ?0.61, ?0.70 and ?0.98, respectively), indicating very high reverse correlation between carvacrol and thymol. In conclusion, the high value of the essential oil compositions in studied S. bachtiarica populations could provide useful information for conservation and selection of cross parents in breeding programs to develop improved cultivars.     

Nanocapsular Dispersion of Thymol for Enhanced Dispersibility and Increased Antimicrobial Effectiveness against Escherichia coli O157:H7 and Listeria monocytogenes in Model Food Systems

Essential oils are marginally soluble in water, making it challenging to evenly disperse them in foods and resulting in an increased tendency to bind with food lipids and proteins, resulting in lowered antimicrobial efficacy. In the current study, free and nano-dispersed (ND) thymol were compared in terms of their antimicrobial efficacies against Escherichia coli O157:H7 ATCC 43889 and 43894 and Listeria monocytogenes strains Scott A and 101 in apple cider and 2% reduced-fat milk. Apple cider was adjusted to pHs 5.5 and 3.5, and antimicrobial tests were performed at 0.3-, 0.5-, 0.75-, and 1.0-g/liter thymol concentrations at 35, 32, 25, and 4°C. Overall, 0.5 and 1.0 g/liter thymol in nano-dispersion and along with free thymol were inhibitory and bactericidal, respectively, against bacterial strains under all treatment conditions. At pH 5.5, 0.5 g/liter ND thymol was bacteriostatic against L. monocytogenes and E. coli for up to 48 h. At pH 3.5, L. monocytogenes controls did not survive beyond 12 h but E. coli survived and was inhibited by 0.5 g/liter ND thymol after 12 and 48 h in apple cider. E. coli strains were significantly sensitive to 4°C and pH 3.5 (P < 0.05). When bacteria were tested in 2% reduced-fat milk at 35 or 32°C, ND and free thymol demonstrated inhibition at 4.5 g/liter. Thus, the current technology seems to be promising and novel, enabling thymol-containing nano-dispersions that are not only transparent but also effective against pathogens in food applications, especially in clear beverages.     

Multiplex PCR for Simultaneous Detection of Bacteria of the Genus Listeria, Listeria monocytogenes, and Major Serotypes and Epidemic Clones of L. monocytogenes

A multiplex PCR assay which combines detection of bacteria of the genus Listeria, Listeria monocytogenes serotypes 1/2a and 4b, and epidemic clones I, II, and III of L. monocytogenes was developed. The assay provides a rapid, reliable, and inexpensive method for screening and subgrouping this important food-borne pathogen.     

Nisin H Is a New Nisin Variant Produced by the Gut-Derived Strain Streptococcus hyointestinalis DPC6484

Accumulating evidence suggests that bacteriocin production represents a probiotic trait for intestinal strains to promote dominance, fight infection, and even signal the immune system. In this respect, in a previous study, we isolated from the porcine intestine a strain of Streptococcus hyointestinalis DPC6484 that displays antimicrobial activity against a wide range of Gram-positive bacteria and produces a bacteriocin with a mass of 3,453 Da. Interestingly, the strain was also found to be immune to a nisin-producing strain. Genome sequencing revealed the genetic determinants responsible for a novel version of nisin, designated nisin H, consisting of the nshABTCPRKGEF genes, with transposases encoded between nshP and nshR and between nshK and nshG. A similar gene cluster is also found in S. hyointestinalis LMG14581. Notably, the cluster lacks an equivalent of the nisin immunity gene, nisI. Nisin H is proposed to have the same structure as the prototypical nisin A but differs at 5 amino acid positions—Ile1Phe (i.e., at position 1, nisin A has Ile while nisin H has Phe), Leu6Met, Gly18Dhb (threonine dehydrated to dehydrobutyrine), Met21Tyr, and His31Lys—-and appears to represent an intermediate between the lactococcal nisin A and the streptococcal nisin U variant of nisin. Purified nisin H inhibits a wide range of Gram-positive bacteria, including staphylococci, streptococci, Listeria spp., bacilli, and enterococci. It represents the first example of a natural nisin variant produced by an intestinal isolate of streptococcal origin.     

The Acid Tolerance Response Alters Membrane Fluidity and Induces Nisin Resistance in Listeria monocytogenes

The ability of L. monocytogenes cells to adapt to a variety of stressors contributes to its growth in a wide range of foods. The present study examines the effect of acid and of the acid tolerance response (ATR) on membrane fluidity and on the organism’s resistance to acid and to the bacteriocin nisin. When ATR was induced in wild-type cells, these cells also became resistant to nisin. ATR(+) cells also had lower membrane rigidities than control ATR(?) cells that had not been subjected to the acid tolerance response. However, cells that were genetically resistant to nisin did not show any significant (P < 0.05) change in rigidity when grown in the presence of nisin. These studies suggest that the use of acid and nisin for L. monocytogenes control in ready-to-eat foods may be compromised if cross-resistance emerges.