Enteritidis PT4 Research Articles

Exploiting antagonistic activity of fruit-derived Lactobacillus to control pathogenic bacteria in fresh cheese and chicken meat

This study assessed the antagonistic activity of fruit-derived lactic acid bacteria (LAB) strains against food-related bacteria and the effects of the highest organic acids LAB producers on the survival of Listeria monocytogenes and Salmonella Enteritidis PT4 in cheese and chicken meat, respectively. The production of organic acids by the Lactobacillus strains in the tested food matrices was also monitored. All tested LAB strains showed antagonistic activity in vitro on the growth of pathogenic or spoiling food-related bacteria, particularly on L. monocytogenes and/or S. Enteritidis PT4, through the action of non-proteinaceous substances. The highest amounts of acetic and lactic acid were detected in cell free culture supernatants of L. paracasei 108 and L. plantarum 201. In “Minas Frescal” cheese, L. plantarum 49 and L. paracasei 108 decreased the counts of L. monocytogenes, and L. plantarum 201 showed bacteriostatic effects on this pathogen over time. L. paracasei 108 decreased the counts of S. Enteritidis PT4 in ground chicken breast; L. plantarum 49 and L. plantarum 201 failed to decrease the counts of this pathogen. Decreases in counts of L. monocytogenes or S. Enteritidis in “Minas Frescal” cheese and ground chicken breast, respectively, were related with increases in lactic and acetic acid contents and decreases in pH values. L. plantarum 49 and L. paracasei 108 could be used as biopreservation tools in cheese and chicken breast meat, respectively.

Influence of storage temperature/time and atmosphere on survival and thermal inactivation of Escherichia coli ATCC 25922 inoculated to almond powder

The purposes of this study were to explore combined effects of storage temperature, time and atmosphere on survival and thermal inactivation of pathogenic Salmonella Enteritidis PT 30 with nonpathogenic E. coli ATCC 25922 as a surrogate inoculated to low-moisture almond powder. A thermal death time test heating block system (TDT-HBS) was used to rapidly determine thermal inactivation of E. coli ATCC 25922. Surviving populations and D75-values of E. coli ATCC 25922 in the almond powder with 6.0% w.b. were determined monthly for 12-month storage under three packaging atmosphere conditions at −20, 4, and 24 °C. The results showed that survival and thermal inactivation curves of E. coli ATCC 25922 fitted well to both Weibull and first-order-kinetic models, respectively. The effects of storage atmosphere on reducing surviving populations and D75-values of E. coli ATCC 25922 became more remarkable as the storage temperature increased. The reductions of 2.55 log CFU/g for surviving populations and 31.2% for D75-values were observed after 12-months storage at 24 °C under modified atmosphere packaging (MAP), and holding for 50.4 min at 75 °C could reach at least 4-log reductions of E. coli ATCC 25922, which are required by the USDA for safe exportation. Storage under MAP at ambient temperature may hold potential to inhibit surviving populations and reduce thermal resistance of S. Enteritidis PT 30 in low-moisture almond powder.

Gene Expression Response of Salmonella enterica Serotype Enteritidis Phage Type 8 to Subinhibitory Concentrations of the Plant-Derived Compounds Trans-Cinnamaldehyde and Eugenol.

Background: Salmonella Enteritidis phage type 8 (PT8) is a major poultry-associated Salmonella strain implicated in foodborne outbreaks in the United States. We previously reported that two plant-derived compounds generally recognized as safe (GRAS), trans-cinnamaldehyde (TC), and eugenol (EG), significantly reduced S. Enteritidis colonization in broiler and layer chickens. To elucidate potential PT8 genes affected by TC and EG during colonization, a whole-genome microarray analysis of the bacterium treated with TC and EG was conducted.Results: S. Enteritidis PT8 was grown in Luria-Bertani broth at 37°C to an OD600 of ~0.5. Subinhibitory concentrations (SICs; concentration that does not inhibit bacterial growth) of TC (0.01%; 0.75 mM) or EG (0.04%; 2.46 mM) were then added to the culture. S. Enteritidis PT8 RNA was extracted before and 30 min after TC or EG addition. Labeled cDNA from three replicate experiments was subsequently hybridized to a microarray of over 99% of S. Enteritidis PT4 genes, and the hybridization signals were quantified. The plant-derived compounds down-regulated (P < 0.005) expression of S. Enteritidis PT8 genes involved in flagellar motility, regulation of the Salmonella Pathogenicity Island 1, and invasion of intestinal epithelial cells. TC and EG also suppressed transcription of genes encoding multiple transport systems and outer membrane proteins. Moreover, several metabolic and biosynthetic pathways in the pathogen were down-regulated during exposure to the plant-derived compounds. Both TC and EG stimulated the transcription of heat shock genes, such as dnaK, dnaJ, ibpB, and ibpA in S. Enteritidis PT8 (P < 0.005). The results obtained from microarray were validated using a quantitative real-time PCR.Conclusion: The plant-derived compounds TC and EG exert antimicrobial effects on S. Enteritidis PT8 by affecting multiple genes, including those associated with virulence, colonization, cell membrane composition, and transport systems.

Inhibitory effect of Nigella sativa oil against Listeria monocytogenes and Salmonella Enteritidis inoculated in minced beef meat

The control of pathogenic bacteria and oxidative factors is important to keep microbiological and physicochemical traits of meat products. In this study, cold-pressed black cumin seed oil (BCSO) was incorporated (at levels of 1, 2, and 4%, w/w) into ground beef meat (GBM) to test the antimicrobial activity of BCSO on the growth inhibition of food-borne pathogens (Listeria monocytogenes Scott A and Salmonella Enteritidis PT4) artificially inoculated (~4.5 log CFU/g) in air-packed GBM during storage at 4 °C for 15 days. BCSO was analyzed for fatty acids, sterols, tocopherols, and thymoquinone using gas chromatography and high-performance liquid chromatography. BCSO induced higher inhibition zone (15–17 mm) against both pathogenic bacteria as compared with the control. The BCSO supplementation retarded the total bacterial count, while the food-borne pathogens were unable to grow in BCSO-supplemented GBM during cold storage for 15 days. After 15 days, S. Enteritidis PT4 was more resistant than L. monocytogenes Scott A in GBM supplemented with 4% BCSO. In general, the shelf life of BCSO-supplemented GBM was extended under refrigerated conditions with low microbial loads. BCSO-supplemented GBM also improved oxidative stability and sodium dodecyl sulfate polyacrylamide gel electrophoresis profile.

Antimicrobial efficacy of cinnamon oil against Salmonella in almond based matrices

Reduced moisture enhances resistance of Salmonella and subsequently reduces the antimicrobial efficacy of thermal treatment. Alternative and supplementary non-thermal intervention methods are urgently needed. In this study, Cinnamonum cassia oil was tested for its antimicrobial effect against outbreak strains Salmonella Enteritidis PT30 and S. Tennessee K4643. Minimal inhibitory concentration and minimal bactericidal concentration for both strains were 0.05% (v/v) and 0.1% (v/v), respectively. Death curves showed that including 0.1% and 0.15% (v/v) C. cassia oil resulted in ∼7 Log reduction of bacteria within 2 h and 1 h, respectively. However, the antimicrobial efficacy of C. cassia oil was reduced when S. Enteritidis PT30 existed in low moisture condition. When S. Enteritidis PT30 was established on almonds/paper discs, 0.4% C. cassia oil resulted in ∼1.7 Log10 CFU/almond or 3.2 Log10 CFU/disc reduction within 2 h at room temperature, respectively. S. Enteritidis PT30 established on both almonds and paper discs were very stable, there was only a 0.80 Log10 CFU/almond and 1.20 Log10 CFU/disc reduction during 9-week and 7-week storage at room temperature, respectively. C. cassia oil intervention increased S. Enteritidis PT30 reduction on both almonds and paper discs during storage with more reduction on paper discs. 0.4% C. cassia oil treatment reduced S. Enteritidis PT30 on paper disc to undetectable level within 4 weeks, but only led to 2 Log10 CFU reduction on almonds, indicating a protection effect from the almond matrix or almond surface components. Additionally, S. Enteritidis PT30 established on paper disc coated with almond surface components exhibited higher resistance to desiccation and C. cassia oil treatment, further demonstrating the protection role of food matrix. In conclusion, C. cassia oil is effective against S. Enteritidis PT30 and S. Tennessee K4643, but its antimicrobial efficacy against the tested Salmonella was compromised by low moisture environment and food matrix.

Changes in thermo-tolerance and survival under simulated gastrointestinal conditions of Salmonella Enteritidis PT4 and Salmonella Typhimurium PT4 in chicken breast meat after exposure to sequential stresses

This study assessed changes in thermo-tolerance and capability to survive to simulated gastrointestinal conditions of Salmonella Enteritidis PT4 and Salmonella Typhimurium PT4 inoculated in chicken breast meat following exposure to stresses (cold, acid and osmotic) commonly imposed during food processing. The effects of the stress imposed by exposure to oregano (Origanum vulgare L.) essential oil (OVEO) on thermo-tolerance were also assessed. After exposure to cold stress (5°C for 5h) in chicken breast meat the test strains were sequentially exposed to the different stressing substances (lactic acid, NaCl or OVEO) at sub-lethal amounts, which were defined considering previously determined minimum inhibitory concentrations, and finally to thermal treatment (55°C for 30min). Resistant cells from distinct sequential treatments were exposed to simulated gastrointestinal conditions. The exposure to cold stress did not result in increased tolerance to acid stress (lactic acid: 5 and 2.5μL/g) for both strains. Cells of S. Typhimurium PT4 and S. Enteritidis PT4 previously exposed to acid stress showed higher (p<0.05) tolerance to osmotic stress (NaCl: 75 or 37.5mg/g) compared to non-acid-exposed cells. Exposure to osmotic stress without previous exposure to acid stress caused a salt-concentration dependent decrease in counts for both strains. Exposure to OVEO (1.25 and 0.62μL/g) decreased the acid and osmotic tolerance of both S. Enteritidis PT4 and S. Typhimurium PT4. Sequential exposure to acid and osmotic stress conditions after cold exposure increased (p<0.05) the thermo-tolerance in both strains. The cells that survived the sequential stress exposure (resistant) showed higher tolerance (p<0.05) to acidic conditions during continuous exposure (182min) to simulated gastrointestinal conditions. Resistant cells of S. Enteritidis PT4 and S. Typhimurium PT4 showed higher survival rates (p<0.05) than control cells at the end of the in vitro digestion. These results show that sequential exposure to multiple sub-lethal stresses may increase the thermo-tolerance and enhance the survival under gastrointestinal conditions of S. Enteritidis PT4 and S. Typhimurium PT4.

Radiofrequency inactivation of Salmonella Enteritidis PT 30 and Enterococcus faecium in wheat flour at different water activities

Salmonella persistence in low-moisture foods creates a significant need for effective pasteurisation processes, but conventional thermal treatments for low-moisture products are challenged by long treatment times and insufficient information on inactivation kinetics. Radiofrequency (RF) heating can reduce heating time and inactivate Salmonella without inducing significant quality damage. The objectives were to study RF heating of organic wheat flour, and evaluate Enterococcus faecium as a surrogate for RF inactivation of Salmonella. Temperature profiles and uniformity of the top and cross-section surface of RF heated flour were obtained with an infrared camera, using different electrode gaps, platforms, and different materials that surrounded the sample to make the electromagnetic field uniform. The flour was inoculated with S. Enteritidis PT 30 or E. faecium, equilibrated to a specific aw, and then RF heated for 8.5 (0.25 aw) or 9 min (0.45 and 0.65 aw) to reach ≈75 °C minimum temperature (no holding time); survivors were then enumerated. The best temperature uniformity was obtained using a 90 mm electrode gap, placing small polystyrene cylinders above and underneath the sample container, and using a platform of polystyrene Petri dishes. Salmonella reduction of 7 log was achieved at 0.45 and 0.65 aw at room temperature, while 5 and 3 log reductions were reached for Salmonella and E. faecium, respectively, at 0.25 aw. These data suggest that RF heating has potential as an inactivation treatment for Salmonella, and that E. faecium is a feasible surrogate to validate the efficacy of RF treatments.

Influence of controlled atmosphere on thermal inactivation of Escherichia coli ATCC 25922 in almond powder

Heat controlled atmosphere (CA) treatments hold potential to pasteurize Salmonella enteritidis PT 30 in almonds. Nonpathogenic Escherichia coli ATCC 25922 was used as a surrogate species of pathogenic Salmonella for validation of thermal pasteurization to meet critical safety requirements. A controlled atmosphere/heating block system (CA-HBS) was used to rapidly determine thermal inactivation of E. coli ATCC 25922. D- and z-values of E. coli ATCC 25922 inoculated in almond powder were determined at four temperatures between 65 °C and 80 °C under different gas concentrations and heating rates. The results showed that D- and z-values of E. coli under CA treatment were significantly (P < 0.05) lower than those under regular atmosphere (RA) treatment at 4 given temperatures. Relatively higher CO2 concentrations (20%) and lower O2 concentrations (2%) were more effective to reduce thermal inactivation time. There were no significant differences in D-values of E. coli when heating rates were above 1 °C/min both in RA and CA treatments. But D-values significantly (P < 0.05) increased under RA treatment and decreased under CA treatment at lower heating rates. Combination of rapid heat and CA treatments could be a promising method for thermal inactivation of S. enteritidis PT 30 in almond powder.

Acyl homoserine lactone-based quorum sensing stimulates biofilm formation by Salmonella Enteritidis in anaerobic conditions.

Quorum sensing regulates a variety of phenotypes in bacteria including the production of virulence factors. Salmonella spp. have quorum sensing systems mediated by three autoinducers (AI-1, AI-2, and AI-3). The AI-1-mediated system is incomplete in that the bacterium relies on the synthesis of signaling molecules by other microorganisms. This study aimed to evaluate the influence of the AI-1 N-dodecanoyl-DL-homoserine lactone (C12-HSL) on the growth, motility, adhesion, and biofilm formation of Salmonella enterica serovar Enteritidis PT4 578 on a polystyrene surface. Experiments were conducted at 37°C in anaerobic tryptone soy broth supplemented with C12-HSL and/or a mixture of four synthetic furanones, at the concentration of 50nM each. The planktonic growth, adhesion, swarming, and twitching motility were not altered in the presence of C12-HSL and/or furanones under anaerobic conditions. However, C12-HSL induced biofilm formation after 36h of cultivation as determined by quantification of biofilm formation, by enumeration of adhered cells to polystyrene coupons, and finally by imaging the presence of multilayered cells on an epifluorescence microscope. When furanones were present in the medium, an antagonistic effect against C12-HSL on the biofilm development was observed. The results demonstrate an induction of biofilm formation in Salmonella Enteritidis by AI-1 under anaerobic conditions. Considering that Salmonella does not produce AI-1 but respond to it, C12-HSL synthesized by other bacterial species could trigger biofilm formation by this pathogen in conditions that are relevant for its pathogenesis.

Novel insights from molecular docking of SdiA from Salmonella Enteritidis and Escherichia coli with quorum sensing and quorum quenching molecules

Quorum sensing is a cell-to-cell communication mechanism leading to differential gene expression in response to high population density. The autoinducer-1 (AI-1) type quorum sensing system is incomplete in Escherichia coli and Salmonella due to the lack of the AI-1 synthase (LuxI homolog) responsible for acyl homoserine lactone (AHL) synthesis. However, these bacteria encode the AHL receptor SdiA (a LuxR homolog) leading to gene regulation in response to AI-1 produced by other bacteria. This study aimed to model the SdiA protein of Salmonella enterica serovar Enteritidis PT4 578 based on three crystallized SdiA structures from Enterohemorrhagic E. coli (EHEC) with different ligands. Molecular docking of these predicted structures with AHLs, furanones and 1-octanoyl-rac-glycerol were also performed. The available EHEC SdiA structures provided good prototypes for modeling SdiA from Salmonella. The molecular docking of these proteins showed that residues Y63, W67, Y71, D80 and S134 are common binding sites for different quorum modulating signals, besides being conserved among other LuxR type proteins. We also show that AHLs with twelve carbons presented better binding affinity to SdiA than AHLs with smaller side chains in our docking analysis, regardless of the protein structures used. Interestingly, the conformational changes provided by AHL binding resulted in structural models with increased affinities to brominated furanones. These results suggest that the use of brominated furanones to inhibit phenotypes controlled by quorum sensing in Salmonella and EHEC may present a good strategy since these inhibitors seem to specifically compete with AHLs for binding to SdiA in both pathogens.

A new repertoire of informations about the quorum sensing system in Salmonella enterica serovar Enteritidis PT4

Salmonella spp are among the main causative agents of foodborne diseases. Some phenotypes associated with increased drug resistance and virulence are regulated by quorum sensing (QS). In the present study, the autoinducer (AI)-1- and -2-mediated QS mechanisms were characterized in Salmonella enterica serovar Enteritidis PT4 for the first time. Salmonella Enteritidis did not produce AI-1. Phylogenetic analysis of nucleotides encoding the SdiA protein, the response regulator of AI-1-mediated QS, and comparative alignment of its amino acids showed that the gene and protein are conserved within the same bacterial genus. Thus, bacteria of the same genus respond to the same AIs. However, this finding did not preclude the possibility that Salmonella Enteritidis might respond to AIs released from bacteria of a different genus, which might confer a competitive advantage to this pathogen. We found that the regulation of AI-2-mediated QS in Salmonella Enteritidis is similar to that in serovar Typhimurium. The elucidation of the AI-1- and AI-2-mediated QS mechanisms in Salmonella Enteritidis will contribute to the development of new control strategies for this pathogen by indicating new targets for antimicrobial drugs.

Assessing the effects of exposure to environmental stress on some functional properties of Bifidobacterium animalis ssp. lactis.

This study assessed the effects of exposing a strain of Bifidobacterium animalis ssp. lactis to acid, bile and osmotic stresses on antagonistic properties, biofilm formation and antibiotic susceptibility/resistance profile. Exposure to each stress factor appeared to have no significant effect on the antagonism against Escherichia coli NCTC 12900 and Salmonella enterica serovar Enteritidis PT4. No suppression in biofilm formation due to exposure to stress was observed. Bile and osmotic stresses resulted in significantly higher biofilm formation. Expression of an exopolysaccharide synthesis gene, gtf 01207, was significantly higher when the B. animalis ssp. lactis strain was exposed to osmotic stress. Susceptibility of the B. animalis ssp. lactis strain to chloramphenicol, erythromycin, ampicillin and vancomycin, and resistance to tetracycline remained unchanged when exposed to each stress. The expression of a tetracycline resistance gene, tet(W), was significantly higher when exposed to each stress. These results may suggest that the potential for the B. animalis ssp. lactis strain to provide probiotic benefit, after exposure to the stressful conditions of the gastrointestinal tract, remains intact.

Total IgA in spleen, bursa and intestine of chickens pretreated with E. faecium AL41 and challenged with Salmonella Enteritidis PT4

The concentration of secretory IgA (sIgA) in intestine flush and solid organs (spleen, bursa) in chickens pretreated with probiotic strain Enterococcus faecium AL41 (EF) and challenged with Salmonella Enteritidis PT4 (SE) was assessed. Forty 1-day-old chickens Cobb 500 were divided into four groups: control, EFAL41, SE and combined EFAL41+SE. The increased concentration of sIgA was determined on day 4 after salmonella infection in the intestine flush in EFAL41group. In combined EFAL41+SE group significant increase of sIgA in the intestine flush was found 7 days post-infection. Pre-treatment of chickens with E. faecium AL41 also showed beneficial effect in bursa in EFAL41 and EFAL41+SE groups. In spleen, total IgA was increased only in SE group. The results demonstrated positive effect of E. faecium AL41 on IgA production in chickens' intestine and solid organs after S. Enteritidis PT4 challenge. Validity of method using for IgA evaluation in solid organ was proved.

Differential protein expression patterns between planktonic and biofilm cells of Salmonella enterica serovar Enteritidis PT4 on stainless steel surface

In the present study, the proteome of a strain of S. enterica serovar Enteritidis PT4, grown either as biofilm on stainless steel surface or as free-floating (planktonic) in Brain Heart (BH) broth, was investigated in order to detect the strong differences in whole-cell protein expression patterns between the two growth styles. The proteins extracted from both types of cells were subjected to 2-D PAGE, followed by in-gel tryptic digestion, extraction, subsequent MALDI-TOF mass spectrometry (MS) analysis and finally database searches for protein identification. Using this approach, 30 proteins were identified as differentially expressed between the two growth modes on an “on-off” basis, that is, proteins that were detected in one case but not in the other. In particular, 20 and 10 proteins were identified in biofilm and planktonic-grown cells, respectively. The group of proteins whose expression was visible only during biofilm growth included proteins involved in global regulation and stress response (ArcA, BtuE, Dps, OsmY, SspA, TrxA, YbbN and YhbO), nutrient transport (Crr, DppA, Fur and SufC), degradation and energy metabolism (GcvT, GpmA, RibB), detoxification (SseA and YibF), DNA metabolism (SSB), curli production (CsgF), and murein synthesis (MipA). To summarize, this study demonstrates that biofilm growth of S. Enteritidis causes distinct changes in protein expression and offers valuable new data regarding some of the proteins presumably involved in this process. The putative role of these proteins in the maintenance of a biofilm community in Salmonella and other bacteria is discussed.

Thermal inactivation of Salmonella enteritidis PT 30 in almond kernels as influenced by water activity.

Salmonellosis outbreaks related to consumption of raw almonds have encouraged the scientific community to study the inactivation kinetics of pathogens in this dry commodity. However, the low moisture content of the product presents a challenge for thermal control, because the time required to achieve the desired thermal inactivation of microorganisms increases sharply with reduced moisture content and water activity. In this study, we explored and modeled the heat inactivation of Salmonella enterica serovar Enteritidis PT 30 in almond cultivar 'Nonpareil' kernel flour at four water activity (a(w)) values (0.601, 0.720, 0.888, and 0.946) using four temperatures for each a(w). The results showed that the inactivation was well fitted by both Weibull distribution (R(2) = 0.93 to 1.00) and first-order kinetics (R(2) = 0.82 to 0.96). At higher a(w) values, the rate of inactivation increased and less time was needed to achieve the required population reduction. These results suggest that, to avoid deterioration of product quality, shorter process times at lower temperatures may be used to achieve desired inactivation levels of Salmonella Enteritidis PT 30 by simply increasing the moisture content of almonds. These goals could be achieved with the use of existing procedures already practiced by the food industry, such as washing or prewetting scalding before heat inactivation.

Complete Genomic Sequence of Salmonella enterica Serovar Enteritidis Phage SE2

Salmonella enterica serovar Enteritidis has remained a major food-borne pathogen in humans. We isolated a virulent S. enterica serovar Enteritidis bacteriophage, SE2, which belongs to the family Siphoviridae. Phage SE2 could lyse S. enterica serovar Enteritidis PT-4, and its virulence was maintained even at ambient temperature. The genomic sequence of phage SE2 was composed of 43,221 bp with close similarity to those of Salmonella phage SETP3 and Salmonella phage SS3e. The strong and stable lytic activity of this phage might enable its use as a therapeutic or biocontrol agent against S. enterica serovar Enteritidis.

HLA-B27 Modulates Intracellular Growth of Salmonella Pathogenicity Island 2 Mutants and Production of Cytokines in Infected Monocytic U937 Cells

Background Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells.Methods/Principal FindingsTo further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis.ConclusionsThe study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis.

Cold Plasma Reduction of Salmonella and Escherichia coli O157:H7 on Almonds Using Ambient Pressure Gases

Contamination of raw nuts, including almonds, is a food safety concern. Cold plasma is a novel antimicrobial intervention that can eliminate foodborne pathogens. The objective of this work was to evaluate the efficacy of rapid cold plasma treatments in eliminating Salmonella and Escherichia coli O157:H7 from dry almonds. Three isolates of Salmonella (S. Anatum F4317, S. Stanley H0558, and S. Enteritidis PT30) and 3 isolates of E. coli O157:H7 (C9490, ATCC 35150, and ATCC 43894) were separately grown and spot-inoculated (10 μL) onto whole almonds and allowed to dry for 10 min. Inoculated almonds were treated with a cold plasma jet, with treatment variables evaluated in a factorial design for each isolate: time, distance, and feed gas. Treatment time was 0 s (control), 10 s, or 20 s. Distance from the emitter was 2, 4, or 6 cm. Feed gas was dry air or nitrogen. After treatment, the almonds were sampled using swabs. Survivors were enumerated on tryptic soy agar (TSA) plates. Cold plasma significantly reduced both pathogens on almonds. The greatest reduction observed was 1.34 log cfu/mL reduction of E. coli O157:H7 C9490 after 20 s treatment at 6 cm spacing. The interaction of treatment time with distance from plasma emitter head was complex, and isolate-dependent. Longer duration of treatment did not always result in enhanced reductions. In general, nitrogen as a feed gas resulted in a reduced antimicrobial efficacy compared to dry air. These results indicate that short pulses of atmospheric pressure cold plasma can significantly reduce Salmonella and E. coli O157:H7 on almonds.

Effect of moisture on salmonella spp. heat resistance in cocoa and hazelnut shells

The heat resistance of food-poisoning outbreak and non-outbreak associated strains of Salmonella (S. Enteritidis, S. Montevideo, S. Napoli, S. Oranienburg, S. Poona, S. Senftenberg and S. Typhimurium) was established in confectionery-related materials such as crushed cocoa bean and hazelnut shells at low moisture contents (≤4% w/w). The two most heat resistant strains in cocoa and hazelnut shells at ca. 4% w/w moisture were S. Oranienburg and S. Enteritidis PT30. Both strains were associated with outbreaks from dried materials. Their D100°C values were ca. 2.5min in crushed cocoa bean shells and 7–11min in crushed hazelnut shells. Addition of moisture to ca. 7% w/w markedly reduced D-values (D80°C of 2–4.5min) for both strains in the two matrices.

Counteracting Recontamination of Pasteurized Milk by Methylated Soybean Protein

Methylated soy protein (MSP) was evaluated as an antimicrobial agent that can counteract the potential post-pasteurization contamination of milk during a 30-day cold storage (4 °C) or 24-h room temperature storage (25 °C) as compared to its native form (SP). SP and MSP were added to buffalo milk at 0.5% (w/v) after pasteurization and before storage. Milk microbiological and chemical analyses were carried out either directly to follow the spontaneous microbial contamination or after artificial contamination with three pathogens (Escherichia coli O157:H7, Listeria monocytogenes Scott A, and Salmonella enterica subsp. enterica serovar Enteritidis PT4). Supplementation of milk samples with MSP (0.5%) significantly (p < 0.05) and considerably inhibited the levels of the spontaneously proliferating bacterial counts, i.e., total viable and Enterobacteriaceae counts were inhibited by about 2.5 log CFU mL−1, while psychrotrophic and pseudomonads counts were inhibited by 1.8 and 1.6 log CFU mL−1, respectively, after 16 days of preservation at 4 °C. Similar trend of effects were also produced after 12–18 h of milk storage at 25 °C. MSP (0.5%) nearly hindered the proliferation of the three inoculated pathogens in pasteurized milk during 16 days of storage at 4 °C or 12–18-h storage at 25 °C. Based on milk acidity, SDS–PAGE pattern, and microbiological analysis, it is concluded that supplementation with MSP (0.5%) can prolong the shelf life of pasteurized milk from 6 to 16 days when stored under cold conditions and from 8 to 18 h under room temperature conditions.