Effects of 1-methylcyclopropene and gaseous ozone on Listeria innocua survival and fruit quality of Granny Smith apples during long-term commercial cold storage.

This study evaluated the impact of 1-methylcyclopropene (1-MCP), an ethylene synthesis inhibitor, followed by long-term commercial cold storage with low-dose gaseous ozone on the microbiological safety and quality of fresh apples. Granny Smith apples were inoculated with or without Listeria innocua, treated with or without 1.0 mg/L 1-MCP for 24 h, then subjected to commercial cold storage conditions including refrigerated air (RA, 0.6 °C, control), controlled atmosphere (CA, 2% O2, 1% CO2, 0.6 °C), and CA with 51-87 µg/L ozone gas for up to 36 weeks. RA storage reduced L. innocua on apples by up to 3.6 log10 CFU/apple. CA had no advantage over RA in controlling Listeria. Continuous ozone gas application resulted in an additional ∼2.0 log10 CFU/apple reduction of L. innocua (total reduction up to 5.7 log10 CFU/apple) and suppressed native bacteria and fungi. Treatment with 1-MCP had a minor impact on survival of L. innocua or background microbiota on apples, while it significantly delayed fruit ripening and reduced the incidence of superficial scald and internal browning. In summary, 1-MCP treatment followed by CA storage with low-dose continuous ozone gas can effectively control Listeria on fresh apples and delay fruit ripening.

Application of an innovative water-assisted ultraviolet C light technology for the inactivation of microorganisms in tomato processing industries.

We aimed to study the efficacy of a water-assisted UVC light device (WUVC) as an innovative clean technology for the disinfection of fresh sound tomatoes and processing wash water and water turbidity was evaluated as a critical parameter. First, wash waters with different turbidities (from 0.4 to 828 NTU) were inoculated with Listeria innocua and treated in the WUVC device at different dosages. Secondly, fresh tomatoes, inoculated with L. innocua and non-inoculated ones, were treated using the WUVC device containing wash water of different turbidities for different times. The reduction of L. innocua populations on wash water and on the surface of tomato was influenced by turbidity; lower reduction values were observed at higher turbidities. Washing tomatoes with tap water with UVC lamps off (control treatment, TW) decreased L. innocua population on the surface of tomatoes but did not eliminate those bacteria that went into the water. Contrarily, when UVC lights were on, L. innocua population in wash water after treatment significantly decreased, those in clean water being the lowest populations. Reductions of native microbiota on the clean water treated with the highest UV-C radiation dose were lower than those obtained when tomatoes were artificially inoculated. We demonstrated that high reductions of L. innocua population on fresh tomatoes could be achieved using the WUVC system but some drawbacks related to the increase of turbidity should be solved for its implementation in real conditions.

Changes of Viscoelastic Properties of Aptamer-Based Sensing Layers Following Interaction with Listeria innocua.

A multiharmonic quartz crystal microbalance (QCM) has been applied to study the viscoelastic properties of the aptamer-based sensing layers at the surface of a QCM transducer covered by neutravidin following interaction with bacteria Listeria innocua. Addition of bacteria in the concentration range 5 × 103-106 CFU/mL resulted in a decrease of resonant frequency and in an increase of dissipation. The frequency decrease has been lower than one would expect considering the dimension of the bacteria. This can be caused by lower penetration depth of the acoustics wave (approximately 120 nm) in comparison with the thickness of the bacterial layer (approximately 500 nm). Addition of E. coli at the surface of neutravidin as well as aptamer layers did not result in significant changes in frequency and dissipation. Using the Kelvin-Voight model the analysis of the viscoelastic properties of the sensing layers was performed and several parameters such as penetration depth, Γ, viscosity coefficient, η, and shear modulus, µ, were determined following various modifications of QCM transducer. The penetration depth decreased following adsorption of the neutravidin layer, which is evidence of the formation of a rigid protein structure. This value did not change significantly following adsorption of aptamers and Listeria innocua. Viscosity coefficient was higher for the neutravidin layer in comparison with the naked QCM transducer in a buffer. However, a further increase of viscosity coefficient took place following attachment of aptamers suggesting their softer structure. The interaction of Listeria innocua with the aptamer layer resulted in slight decrease of viscosity coefficient. The shearing modulus increased for the neutravidin layer and decreased following aptamer adsorption, while a slight increase of µ was observed after the addition of Listeria innocua.

Flow-Through Electrochemical Biosensor for the Detection of Listeria monocytogenes Using Oligonucleotides.

Consumption of food contaminated by Listeria monocytogenes can result in Listeriosis, an illness with hospitalization rates of 94% and mortality rates up to 30%. As a result, U.S. regulatory agencies governing food safety retain zero-tolerance policies for L. monocytogenes. However, detection at such low concentrations often requires strategies such as increasing sample size or culture enrichment. A novel flow-through immunoelectrochemical biosensor has been developed for Escherichia coli O157:H7 detection in 1 L volumes without enrichment. The current work further augments this biosensor's capabilities to (1) include detection of L. monocytogenes and (2) accommodate genetic detection to help overcome limitations based upon antibody availability and address specificity errors in phenotypic assays. Herein, the conjugation scheme for oligo attachment and the conditions necessary for genetic detection are laid forth while results of the present study demonstrate the sensor's ability to distinguish L. monocytogenes DNA from L. innocua with a limit of detection of ~2 × 104 cells/mL, which agrees with prior studies. Total time for this assay can be constrained to <2.5 h because a timely culture enrichment period is not necessary. Furthermore, the electrochemical detection assay can be performed with hand-held electronics, allowing this platform to be adopted for near-line monitoring systems.

Differential Modulation of Listeria monocytogenes Fitness, In Vitro Virulence, and Transcription of Virulence-Associated Genes in Response to the Presence of Different Microorganisms.

Interactions between Listeria monocytogenes and food-associated or environmental bacteria are critical not only for the growth but also for a number of key biological processes of the microorganism. In this regard, limited information exists on the impact of other microorganisms on the virulence of L. monocytogenes In this study, the growth of L. monocytogenes was evaluated in a single culture or in coculture with L. innocua, Bacillus subtilis, Lactobacillus plantarum, or Pseudomonas aeruginosa in tryptic soy broth (10°C/10 days and 37°C/24 h). Transcriptional levels of 9 key virulence genes (inlA, inlB, inlC, inlJ, sigB, prfA, hly, plcA, and plcB) and invasion efficiency and intracellular growth in Caco-2 cells were determined for L. monocytogenes following growth in mono- or coculture for 3 days at 10°C or 9 h at 37°C. The growth of L. monocytogenes was negatively affected by the presence of L. innocua and B. subtilis, while the effect of cell-to-cell contact on L. monocytogenes growth was dependent on the competing microorganism. Cocultivation affected the in vitro virulence properties of L. monocytogenes in a microorganism-specific manner, with L. innocua mainly enhancing and B. subtilis reducing the invasion of the pathogen in Caco-2 cells. Assessment of the mRNA levels of L. monocytogenes virulence genes in the presence of the four tested bacteria revealed a complex pattern in which the observed up- or downregulation was only partially correlated with growth or in vitro virulence and mainly suggested that L. monocytogenes may display a microorganism-specific transcriptional response.IMPORTANCEListeria monocytogenes is the etiological agent of the severe foodborne disease listeriosis. Important insight regarding the physiology and the infection biology of this microorganism has been acquired in the past 20 years. However, despite the fact that L. monocytogenes coexists with various microorganisms throughout its life cycle and during transmission from the environment to foods and then to the host, there is still limited knowledge related to the impact of surrounding microorganisms on L. monocytogenes' biological functions. In this study, we showed that L. monocytogenes modulates specific biological activities (i.e., growth and virulence potential) as a response to coexisting microorganisms and differentially alters the expression of virulence-associated genes when confronted with different bacterial genera and species. Our work suggests that the interaction with different bacteria plays a key role in the survival strategies of L. monocytogenes and supports the need to incorporate biotic factors into the research conducted to identify mechanisms deployed by this organism for establishment in different environments.

Listeria innocua Dps as a nanoplatform for bioluminescence based photodynamic therapy utilizing Gaussia princeps luciferase and zinc protoporphyrin IX.

Listeria innocua DNA binding protein from starved cells (LiDps) belongs to the ferritin family and provides a promising self-assembling spherical 12-mer protein scaffold for the generation of functional nanomaterials. We report the creation of a Gaussia princeps luciferase (Gluc)-LiDps fusion protein, with chemical conjugation of Zinc (II)-protoporphyrin IX (ZnPP) to lysine residues on the fusion protein (giving Gluc-LiDps-ZnPP). The Gluc-LiDps-ZnPP conjugate is shown to generate reactive oxygen species (ROS) via Bioluminescence Resonance Energy Transfer (BRET) between the Gluc (470-490 nm) and ZnPP. In vitro, Gluc-LiDps-ZnPP is efficiently taken up by tumorigenic cells (SKBR3 and MDA-MB-231 breast cancer cells). In the presence of coelenterazine, this construct inhibits the proliferation of SKBR3 due to elevated ROS levels. Following exposure to Gluc-LiDps-ZnPP, migration of surviving SKBR3 cells is significantly suppressed. These results demonstrate the potential of the Gluc-LiDps-ZnPP conjugate as a platform for future development of an anticancer photodynamic therapy agent.

Strawberry sanitization by peracetic acid washing and its effect on fruit quality.

The risk posed by outbreaks associated with strawberries together with the safety issues of by-products from chlorine disinfection in the fruit industry has led to a search for alternative sanitizers. The disinfection capacity of peracetic acid (PA) at three concentrations (20, 40 and 80 ppm) and washing times (1 and 2 min) was compared to sodium hypochlorite (200 ppm) (NaClO) treatments and a water control, and its influence on the physico-chemical, biochemical and nutritional quality of strawberries was also studied. Counts on total aerobic mesophilic microorganisms were comparable between NaClO and PA. For yeasts and molds, only NaClO and 80 ppm PA reduced contamination in washing water, but no differences wereobserved in strawberries. Artificially inoculated L.innocua was reduced by at least 4 log cfu/g in strawberry by all the PA treatments, except at 20 ppm PA for 1 min. Total soluble solids, pH, titratable acidity, antioxidant activity and total phenolic content values were maintained after all treatments. Only anthocyanin content was affected. Treatments of 20 and 40 ppm PA did not significantly affect fruit color, and there were no losses on strawberry firmness. PA, as a GRAS substance that has shown potential to reduce microorganisms present in strawberries without any major physicochemical or sensorial alteration, could be a suitable alternative to chlorine disinfection.

Coresistance to Benzalkonium Chloride Disinfectant and Heavy Metal Ions in Listeria monocytogenes and Listeria innocua Swine Isolates from China.

The development of coresistance to disinfectants and heavy metals contributes to the fitness of Listeria spp. in foods or food processing environments, where life-threatening Listeria monocytogenes coexist and coevolve with other Listeria spp. Despite extensive research on L. monocytogenes, coresistance to disinfectants and heavy metals is less documented for other Listeria spp. In this study, we screened 30 L. monocytogenes and 27 Listeria innocua isolates recovered from 273 swine samples for resistance to quaternary ammonium compound benzalkonium chloride (BC) and to heavy metals cadmium (Cd) and arsenic (As). Moreover, we evaluated the potential mechanisms of resistance by detecting the efflux pump activity in BC resistance and the presence of resistance determinants. The average minimum inhibitory concentrations of BC in L. innocua (10.7 ± 2.0) were significantly higher than that in L. monocytogenes (6.9 ± 3.7) (p < 0.05). Resistance to BC and heavy metals was correlated, where all BC-resistant L. innocua and As-resistant L. monocytogenes isolates were coresistant to BC and Cd. Twenty percent and 66.7% of BC resistance in L. monocytogenes and L. innocua were related to reserpine-associated efflux pumps, whereas all cases of BC resistance were related to carbonyl cyanide 3-chlorophenylhydrazone-associated efflux pumps. The cadA1 and cadA2 genes were present in Cd-resistant isolates but not in Cd-sensitive isolates, and cadA3 was undetectable in all isolates examined. cadA4 conferring lower level of Cd resistance was copresent with arsA1 and arsA2 in the Cd-resistant and As-susceptible L. monocytogenes isolate LM3. Our findings suggest that swine serves as a reservoir for developing resistance to disinfectant and heavy metals in L. monocytogenes and L. innocua, which share common resistance mechanisms such as efflux pumps and resistance genes. This work provides new insight into the coresistance events of other Listeria as a potential contributor of the resistance in L. monocytogenes.

Synthesis and antibacterial activity of new peptides from Alfalfa RuBisCO protein hydrolysates and mode of action via a membrane damage mechanism against Listeria innocua.

In this work we evaluated the mode of action of six new synthesized peptides (Met-Asp-Asn; Glu-leu-Ala-Ala-Ala-Cys; Leu-Arg-Asp-Asp-Phe; Gly-Asn-Ala-Pro-Gly-Ala-Val-Ala; Ala-Leu-Arg-Met-Ser-Gly and Arg-Asp-Arg-Phe-Leu), previously identified, from the most active peptide fractions of RuBisCO peptic hydrolysate against Listeria innocua via a membrane damage mechanism. Antibacterial effect and the minimum inhibitory concentrations (MIC) of these peptides were evaluated against six strains and their hemolytic activities towards bovine erythrocytes were determined. Prediction of the secondary structure of peptides indicated that these new antibacterial peptides are characterized by a short peptide chains (3-8 amino acid) and a random coli structure. Moreover, it was observed that one key characteristic of antibacterial peptides is the presence of specific amino acids such as cysteine, glycine, arginine and aspartic acid. In addition the determination of the extracellular potassium concentration revealed that treatment with pure RuBisCO peptides could cause morphological changes of L. innocua and destruction of the cell integrity via irreversible membrane damage. The results could provide information for investigating the antibacterial model of antibacterial peptides derived from RuBisCO protein hydrolysates.

Survival of Listeria innocua on Fuji apples under commercial cold storage with or without low dose continuous ozone gaseous.

This study evaluated the fate of Listeria innocua, a non-pathogenic species closely related to Listeria monocytogenes, on Fuji apple fruit surfaces during commercial cold storage with and without continuous low doses of gaseous ozone. Unwaxed Fuji apples of commercially acceptable maturity were inoculated with 6.0-7.0 Log10 CFU L. innocua/apple, and subjected to refrigerated air (RA, 33 °F), controlled atmosphere (CA, 33 °F, 2% O2, 1% CO2), or CA with low doses of ozone gas (50.0 -87.0 ppb ) storage in a commercial facility for 30 weeks. A set of uninoculated apples was simultaneously subjected to the above storage conditions for total plate count and yeasts and molds enumeration. L. innocua survival under RA and CA storage was similar, which led to 2.5-3.0 Log10 CFU/apple reduction during storage. Continuous gaseous ozone application decreased L. innocua population on Fuji apples to ∼1.0 Log10 CFU/apple after 30-week storage, and suppressed apple native flora. CA storage delayed apple fruit ripening through reduction of apple firmness and titratable acidity loss, and low dose gaseous ozone application had no negative influence on apple visual quality, including both external and internal disorders. In summary, L. innocua decreased on Fuji apple surfaces during commercial long-term RA and CA storage. Ozone gas has the potential to be used as a supplemental intervention method to control Listeria spp. and to ensure fresh apple safety.

Antibiofilm Effect of Poly(Vinyl Alcohol-co-Ethylene) Halamine Film against Listeria innocua and Escherichia coli O157:H7.

Bacterial biofilm formation is linked to several infections and foodborne disease outbreaks. To address this challenge, there is an unmet need to develop rechargeable antimicrobial materials that can provide continuous sanitation of contact surfaces, especially in the food industry. This study was aimed at evaluating a novel rechargeable antimicrobial polymer formed using poly(vinyl alcohol-co-ethylene) (PVA-co-PE) with halamine functionality to prevent biofilm formation with repeated exposure to high loads of bacteria and organic content and also to aid in inactivation of preformed biofilms upon contact with this novel material. The antibiofilm activity of this rechargeable antimicrobial material was evaluated using a combination of fluorescence and scanning electron microscopy techniques and biofilm metabolic activity analyses. The results determined on the basis of imaging and metabolic activity measurements demonstrated that halamine-functionalized polymer films significantly reduced Listeria innocua and Escherichia coli O157:H7 biofilm formation. This novel polymeric material maintained its antibiofilm activity with repeated cycles of extended exposure to high levels of bacterial load. These polymeric films were recharged using bleach and cleaned using mechanical sonication after each cycle of extended incubation with bacteria. Halamine-functionalized polymeric material also exhibited significant antibacterial activity against preformed biofilms on a model surface. In summary, our results demonstrate the potential of this antimicrobial material to provide continuous sanitation of surfaces and applications for inactivating preformed biofilms without extensive use of resources, including water and heat. This polymeric material may be used as a replacement for existing polymeric materials or as a coating on diverse materials.IMPORTANCE Conventional sanitizers can have limited efficacy in inactivating biofilms in areas with limited accessibility and buildup of organic biomass. Furthermore, none of the current approaches provide continuous sanitation of surfaces. There is a significant unmet need to develop and validate materials that can prevent biofilm formation as well as inactivate preformed biofilms. In this study, the efficacy of a copolymer film containing N-halamine against biofilms of L. innocua and E. coli O157:H7 was evaluated. The polymer film showed strong inhibitory activity against pregrown biofilm or prevented the growth of a new biofilm. The polymer film also maintained its antibiofilm activity after multiple cycles of exposure to high titers of bacterial load with recharging of the polymer film using bleach at intermediate steps between the cycles. Overall, the results demonstrate the potential of a novel antimicrobial material to inhibit and treat biofilms in food industry applications.

Comparison of Listeria monocytogenes recoveries from spiked mung bean sprouts by the enrichment methods of three regulatory agencies.

Three selective enrichment methods, the United States Food and Drug Administration's (FDA method), the United States Department of Agriculture Food Safety Inspection Service's (USDA method), and the EN ISO 11290-1 standard method, were assessed for their suitability for recovery of Listeria monocytogenes from spiked mung bean sprouts. Three parameters were evaluated; the enrichment L. monocytogenes population from singly-spiked sprouts, the enrichment L. monocytogenes population from doubly-spiked (L. monocytogenes and Listeria innocua) sprouts, and the population differential resulting from the enrichment of doubly-spiked sprouts. Considerable L. monocytogenes inter-strain variation was observed. The mean enrichment L. monocytogenes populations for singly-spiked sprouts were 6.1 ± 1.2, 4.9 ± 1.2, and 6.9 ± 2.3 log CFU/mL for the FDA, USDA, and EN ISO 11290-1 methods, respectively. The mean L. monocytogenes populations for doubly-spiked sprouts were 4.7 ± 1.1, 5.5 ± 1.3, and 4.6 ± 1.4 log CFU/mL for the FDA, USDA, and ISO 11290-1 enrichment methods, respectively. The corresponding mean population differentials were 2.8 ± 1.1, 3.3 ± 1.3, and 3.6 ± 1.4 Δlog CFU/mL for the same three enrichment methods, respectively. The presence of L. innocua and resident microorganisms on the sprouts negatively impacted final levels of L. monocytogenes with all three enrichment methods.

Fate of Listeria innocua during production and ripening of smeared hard cheese made from raw milk.

The fate of 2 different Listeria innocua strains was analyzed during the production and ripening of smeared raw milk Greyerzer cheese (Gruyère). These strains were used as surrogates for the pathogenic Listeria monocytogenes, as they are physiologically very similar. Bacterial cells were added to the cheese milk at levels of 105 cfu/mL. During the first 24 h of cheese making, the number of the test strains decreased to a level of below 102 cfu/g. Obviously, the cooking temperature of 56°C and the subsequent slight temperature decrease to 50°C within 70 min contributed to a distinct reduction of Listeria counts. The counts in the cheese cores did not exceed 103 cfu/g within 12 wk of cheese ripening and Listeria was not detectable after 24 wk. In contrast to the cores of the cheeses of the 4 batches in this study, their rinds always contained a high listerial load of approximately 106 to 108 cfu/g throughout the entire ripening period. The smeared surface showed an increase of pH to alkaline values, corresponding to smear microbiota development. Coryneforms and Staphylococcus counts were stable at >107 cfu/cm2 over 175 d, whereas yeast counts decreased to about 105 cfu/cm2 at the end of ripening. The study shows that the smear culture had no noticeable anti-listerial potential. When removing the rind or portioning such smeared cheese loaves with a cutting device, a postprocess contamination of the core might occur, thus presenting a major hygienic risk.

Antimicrobial Resistance Profiles of Listeria monocytogenes and Listeria innocua Isolated from Ready-to-Eat Products of Animal Origin in Spain.

The objective of this work was to investigate the antimicrobial resistance in Listeria spp. isolated from food of animal origin. A total of 50 Listeria strains isolated from meat and dairy products, consisting of 7 Listeria monocytogenes and 43 Listeria innocua strains, were characterized for antimicrobial susceptibility against nine antimicrobials. The strains were screened by real-time PCR for the presence of antimicrobial resistance genes: tet M, tet L, mef A, msr A, erm A, erm B, lnu A, and lnu B. Multidrug resistance was identified in 27 Listeria strains, 4 belonging to L. monocytogenes. Resistance to clindamycin was the most common resistance phenotype and was identified in 45 Listeria strains; the mechanisms of resistance are still unknown. A medium prevalence of resistance to tetracycline (15 and 9 resistant and intermediate strains) and ciprofloxacin (13 resistant strains) was also found. Tet M was detected in Listeria strains with reduced susceptibility to tetracycline, providing evidence that both L. innocua and L. monocytogenes displayed acquired resistance. The presence of antimicrobial resistance genes in L. innocua and L. monocytogenes indicates that these genes may be transferred to commensal and pathogenic bacteria via the food chain; besides this, antibiotic resistance in L. monocytogenes could compromise the effective treatment of listeriosis in humans.

Inhibitory Effect of Lactococcin BZ Against Listeria innocua and Indigenous Microbiota of Fresh Beef.

In this study, the effect of lactococcin BZ on microbiological quality of fresh beef is investigated. For this purpose, the meat samples were treated with various amounts of lactococcin BZ (200-2500 AU/mL), a bacteriocin produced by Lactococcus lactis spp. lactis BZ, and kept at 4-5 °C for 12 days. During storage, the microbiological properties of the meat samples with or without lactococcin BZ were determined. Inhibitory effect of lactococcin BZ depended on its amount. The higher the amount of lactococcin BZ, the higher the inhibitory activity. Treatment with lactococcin BZ at the level of 2500 AU/mL resulted in 4.87, 3.50 and 3.94 log cycle decrease in the counts of mesophilic, psychrotrophic and lactic acid bacteria, respectively, and 1.90·104 and 1.04·102 CFU/g reduction in coliform and faecal coliform bacteria, respectively, at the end of storage as compared to their initial numbers in the control sample. However, the counts of these bacteria in control samples increased during storage. Also, lactococcin BZ at 1600 AU/mL showed very strong antilisterial effect against Listeria innocua in fresh meat and reduced the cell numbers from 6.04 log CFU/g to undetectable level on the 6th day of storage. In conclusion, lactococcin BZ has a potential use as a biopreservation agent to improve safety and shelf life of raw beef.

Sublethal injury and virulence changes in Listeria monocytogenes and Listeria innocua treated with antimicrobials carvacrol and citral.

The aim of this study was to evaluate the effect of two antimicrobial substances, carvacrol and citral, on Listeria monocytogenes and Listeria innocua cells, as well as possible virulence changes in injured cells, using Caenorhabditis elegans as a model test. The results indicated that the percentage of sublethal damage was higher in L. monocytogenes than in L. innocua. The results of the study carried out by using C. elegans indicated that C. elegans fed in a lawn of L. monocytogenes previously treated with carvacrol showed a loss in life span (p ≤ 0.05) as compared with L. monocytogenes treated with citral, Escherichia coli OP50 as a negative control, and treated and untreated L. innocua. Egg laying was also affected: worms fed in a lawn of treated and untreated L. monocytogenes laid fewer eggs than those fed in a lawn of treated and untreated L. innocua or fed with OP50 as a negative control. Worms fed in a lawn of treated and untreated L. innocua also laid fewer eggs than those fed with OP50 as a negative control. A phenotype named bag of worms and an undescribed new one, "vulva inflammation", were also observed.

Antimicrobial Activity of Rhoeo discolor Phenolic Rich Extracts Determined by Flow Cytometry.

Traditional medicine has led to the discovery of important active substances used in several health-related areas. Phytochemicals in Rhoeo discolor extracts have proven to have important antimicrobial activity. In the present study, our group determined the antimicrobial effects of extracts of Rhoeo discolor, a plant commonly used in Mexico for both medicinal and ornamental purposes. We evaluated the in vitro activity of phenolic rich extracts against specifically chosen microorganisms of human health importance by measuring their susceptibility via agar-disc diffusion assay and flow cytometry: Gram-positive Listeria innocua and Streptococcus mutans, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and lastly a fungal pathogen Candida albicans. Ten different extracts were tested in eight different doses on all the microorganisms. Analytical data revealed a high content of phenolic compounds. Both agar-disc diffusion assay and flow cytometry results demonstrated that Pseudomonas aeruginosa was the least affected by extract exposure. However, low doses of these extracts (predominantly polar), in a range from 1 to 4 µg/mL, did produce a statistically significant bacteriostatic and bactericidal effect on the rest of the microorganisms. These results suggest the addition of certain natural extracts from Rhoeo discolor could act as antibacterial and antimycotic drugs or additives for foods and cosmetics.

Effect of citral and carvacrol on the susceptibility of Listeria monocytogenes and Listeria innocua to antibiotics.

UNLABELLED: The aim of this study was to evaluate the antibiotic susceptibility of Listeria innocua (L. innocua) and Listeria monocytogenes (L. monocytogenes) cells in the presence of citral and carvacrol at sublethal concentrations in an agar medium. The presence of terpenes in the L. monocytogenes and L. innocua culture medium provided a reduction in the minimal inhibitory concentration (MIC) of all the antibiotics tested. These effects were dependent on the concentration of terpenes present in the culture medium. The combination of citral and carvacrol potentiated antibiotic activity by reducing the MIC values of bacitracin and colistin from 32.0 and 128.0 µg ml⁻¹ to 1.0 and 2.0 µg ml⁻¹, respectively. Thus, both Listeria species became more susceptible to these drugs. In this way, the colistin and bacitracin resistance of L. monocytogenes and L. innocua was reversed in the presence of terpenes. Results obtained in this study show that the phytochemicals citral and carvacrol potentiate antibiotic activity, reducing the MIC values of cultured L. monocytogenes and L. innocua. SIGNIFICANCE AND IMPACT OF THE STUDY: Phytochemicals citral and carvacrol potentiate antibiotic activity of erythromycin, bacitracin and colistin by reducing the MIC values of cultured Listeria monocytogenes and Listeria innocua. This effect in reducing the MIC values of the antibiotics tested in both micro-organisms was increased when natural antimicrobials were combined. This finding indicated that the combination among terpenes and antibiotic may contribute in reducing the required dosage of antibiotics due to the possible effect of terpenes on permeation barrier of the micro-organism cell membrane.

Use of the modified Gompertz equation to assess the Stevia rebaudiana Bertoni antilisterial kinetics.

In order to assess the antibacterial activity of Stevia rebaudiana Bertoni (Stevia), Listeria innocua growth was characterized at 37 °C, in reference medium supplemented with a leaf infusion, a crude extract, and a steviol glycosides purified extract. Experimental data were fitted to the modified Gompertz model and the antibacterial activity of Stevia was determined based on the lag time (λ) and the maximum growth rate (µmax) reached, depending on the incubation conditions. As the leaf infusion showed the most marked elongation of λ and the most marked µmax reduction, its antimicrobial effect was evaluated at different concentrations, at 37, 22 and 10 °C. According to the results obtained, in general, the lower the temperature or the higher the Stevia concentration, the longer the λ and the lower the µmax, statistically significant being the effect of reducing temperature from 37 or 22 to 10 °C, the effect of increasing Stevia concentration from 0 or 0.5 to 1.5 or 2.5% (w/v), at 37 °C, and the elongation of λ observed in presence of 1.5 and 2.5% (w/v) of Stevia, at 22 °C. These results show that Stevia could be a bacterial growth control measure if a cold chain failure occurs.

Antimicrobial resistance of Listeria monocytogenes and Listeria innocua from meat products and meat-processing environment.

A total of 336 Listeria isolates from ready-to-eat (RTE) meat products and meat-processing environments, consisting of 206 Listeria monocytogenes, and 130 Listeria innocua isolates, were characterized by disc diffusion assay and minimum inhibitory concentration (MIC) values for antimicrobial susceptibility against twenty antimicrobials. Resistance to one or two antimicrobials was observed in 71 L. monocytogenes isolates (34.5%), and 56 L. innocua isolates (43.1%). Multidrug resistance was identified in 24 Listeria isolates, 18 belonging to L. innocua (13.9%) and 6 to L. monocytogenes (2.9%). Oxacillin resistance was the most common resistance phenotype and was identified in 100% Listeria isolates. A medium prevalence of resistance to clindamycin (39.3% isolates) and low incidence of resistance to tetracycline (3.9% isolates) were also detected. Listeria isolates from RTE meat products displayed higher overall antimicrobial resistance (31.3%) than those from the environment (13.4%). All the strains assayed were sensitive to the preferred antibiotics used to treat listeriosis. Results showed that although antimicrobial resistance in L. monocytogenes still occurs at a low prevalence, L. innocua can form a reservoir of resistance genes which may transfer between bacterial species, including transference to organisms capable of causing disease in humans.