Effect of microbial loading on the efficiency of cold atmospheric gas plasma inactivation of Salmonella enterica serovar Typhimurium

In recent years the application of cold atmospheric gas plasma (CAP) aimed at the removal of microbial contamination from fresh and minimally processed food has received increased attention. For CAP to be successfully adopted by the food production industry, factors which affect its potential for microbial inactivation must be evaluated. In this study, we examined the effects of initial microbial concentration, present on filter discs, on the inactivation of Salmonella enterica serovar Typhimurium (S. Typhimurium) with nitrogen CAP. It was found that the rate of inactivation of S. Typhimurium is inversely proportional to initial bacterial concentration, with the D-value observed at the highest cell concentration assayed (10⁸ CFU/filter) being 14 fold higher than seen at the lowest starting concentration (10⁵ CFU/filter). Addition of increasing concentrations of Pseudomonas fluorescens cells to a Salmonella population of 10⁵ CFU/filter resulted in an exponential decrease in the rate of killing of the Salmonella cells. However, whilst the addition of heat-killed S. Typhimurium cells to 10⁵ CFU/filter live S. Typhimurium cells resulted in a significant decrease in the killing rate, this effect was dose independent. This suggests that although biomass plays a role in the protection against CAP inactivation seen at high cell densities, dead cells and their components released during the heating period are not as effective as viable cells. Fluorescence microscopy showed that, unlike the single dispersed cells observed at low cell densities, at higher cell densities bacteria were present in a multilayered structure. This phenomenon could explain the reduced inactivation by the plasma, since the top layer may present a physical barrier that protects underlying cells. In conclusion, this work clearly shows a link between bacterial cell density and the efficacy of CAP inactivation, making an important contribution to the understanding of this alternative food processing technology, which should be taken into account in both further studies and in the practical application of this technique to the food industry.     

Emergence of pulsed electric fields resistance in Salmonella enterica serovar Typhimurium SL1344

In this investigation we selected and isolated a culture derived from Salmonella enterica serovar Typhimurium SL1344 with stable increased resistance to pulsed electric fields (PEF) after repeated rounds of PEF treatment and outgrowth of survivors. The resulting culture showed a higher resistance to PEF treatments under different treatment conditions. The acquisition of PEF resistance was only observed in stationary phase cells. The cytoplasmic membrane of the resistant variant showed a higher resilience against PEF treatments, since a lower permeabilization degree was observed after PEF treatments, in comparison to the parental strain. Resistance to PEF was also accompanied by a higher tolerance to acidic pH, hydrogen peroxide and ethanol, but not to heat. The occurrence of a PEF resistant variant in S. enterica serovar Typhimurium SL1344 emphasizes the need to further study the mechanisms of inactivation and resistance by PEF for an adequate design of safe treatments.     

Kinetics of growth and inactivation of Salmonella enterica serotype Typhimurium DT104 in pasteurised liquid egg products

The potential impact of post-pasteurisation contamination of liquid egg products with the multi-antibiotic resistant pathogen Salmonella enterica serotype Typhimurium definitive type 104 (DT104) was assessed by determining the viability of this bacterium in whole egg, albumen and 10% w/w sugared and salted yolk incubated at 4–42 °C. Results indicated that populations of S. Typhimurium DT104 were slowly inactivated in all four products when stored at 4 °C. However, based on the typical shelf-lives of cold-stored liquid egg, less than 0.6 log-kill would be achieved in those products prior to their use. Incubation at temperatures pertaining to abuse situations (10, 15, 20 and 25 °C) revealed an increasing potential for growth of S. Typhimurium DT104 in whole egg, albumen and sugared yolk, as indicated by trends in growth rate, lag duration and maximum population density. At even higher temperatures (30, 37 and 42 °C), growth rates of S. Typhimurium DT104 in whole egg and sugared yolk continued to increase. The same was true for S. Typhimurium DT104 in albumen except that growth was not observed at 42 °C and instead populations were inactivated within 30 h. At no temperature tested was S. Typhimurium DT104 able to grow in salted yolk. The influence of these growth and inactivation patterns on the risk of salmonellosis in relation to product type and storage temperature is discussed.     

Differences in biofilm formation of produce and poultry Salmonella enterica isolates and their persistence on spinach plants

Spinach plants were irrigated biweekly with water containing 2.1 log CFU Salmonella/100 ml water (the maximum Escherichia coli MPN recommended by the Leafy Greens Marketing Agreement; LGMA), or 4.1 CFU Salmonella/100 ml water to determine Salmonella persistence on spinach leaves. Green Fluorescent protein expressing Salmonella were undetectable by most-probable number (MPN) at 24 h and 7 days following each irrigation event. This study indicates that Salmonella are unlikely to persist on spinach leaves when irrigation water is contaminated at a level below the LGMA standards. In a parallel study, persistence of Salmonella isolated from poultry or produce was compared following biweekly irrigation of spinach plants with water containing 6 log CFU Salmonella/100 ml. Produce Salmonella isolates formed greater biofilms on polystyrene, polycarbonate and stainless steel surfaces and persisted at significantly higher numbers on spinach leaves than those Salmonella from poultry origin during 35 days study. Poultry Salmonella isolates were undetectable (<1 log CFU/g) on spinach plants 7 days following each irrigation event when assayed by direct plating. This study indicates that Salmonella persistence on spinach leaves is affected by the source of contamination and the biofilm forming ability of the strain.     

The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major cause of foodborne salmonellosis. Rapid, efficient and accurate methods for identification are required to track specific strains of S. Enteritidis during outbreaks of human salmonellosis. By exploiting the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we previously developed a powerful sequence-based subtyping approach, designated CRISPR-MVLST. To substantiate the applicability of CRISPR-MVLST, we analyzed a broad set of S. Enteritidis isolates collected over a six-year period. Among 141 isolates we defined 22 Enteritidis Sequence Types (ESTs), the majority of which were novel. Notably, strains exhibiting the common PFGE pattern, JEGX01.0004 (characteristic of ∼40% of S. Enteritidis isolates in the United States), were separated into twelve distinct sequence types. Conversely, isolates of EST4, the most predominant EST we observed, comprised eight different PFGE patterns. Importantly, we showed that some genotypes that were previously associated with the food supply chain at the farm level have now been identified in clinical samples.     

Introduction of new multidrug-resistant Salmonella enterica strains into commercial dairy herds

A longitudinal observational study of 59 dairy herds was conducted in Washington State to estimate the rate of introduction of new multidrug-resistant (MDR) Salmonella enterica strains onto commercial dairy herds. Samples were collected on these herds over 7 visits separated by intervals of 2 to 4 mo over a period of 15 to 21 mo. Samples were cultured for Salmonella spp. and serogroup, serovar, and antimicrobial susceptibility patterns were identified for MDR Salmonella isolates. Fingerprinting generated by pulsed-field gel electrophoresis (PFGE) using XbaI restriction enzyme digestion generated genotyping profiles for all MDR isolates identified in the study. The rate of new MDR Salmonella strain introduction was 0.9 per herd-year (95% confidence interval: 0.6-1.4). The rates for the most commonly introduced MDR Salmonella serovars were 0.4/herd-year for Typhimurium, 1.2/herd-year for Newport, and 0.1/herd-year for Dublin. Thirty-three of 59 herds (56%) had at least one new MDR Salmonella introduction during the study period. The number of new MDR Salmonella strains acquired by dairy herds ranged from zero to 8. Thirteen of the 59 herds had a history of clinical salmonellosis. Among these 13 herds, 6 herds acquired new MDR Salmonella strains, although these strains were different than historical clinical strains. These data indicate that acquisition of new MDR Salmonella strains by dairy herds was a common event in participating herds, although the number of strains introduced varied greatly among herds.     

Use of high-concentration-short-time chlorine dioxide gas treatments for the inactivation of Salmonella enterica spp. inoculated onto Roma tomatoes

Salmonella outbreaks have been recently linked to the consumption of fresh tomatoes. Thus, there is a need to develop systems that reduce the risk of microbial contamination to increase product shelf-life and keep fresh fruit attributes. The objectives of this study were to evaluate high-concentration-short-time chlorine dioxide gas treatments effects on Salmonella-inoculated Roma tomatoes and determine the optimal treatment conditions for microbial inactivation and shelf-life extension. Effects of ClO₂ concentration (2, 5, 8 and 10 mg/l) and exposure time (10, 30, 60, 120 and 180 s) on inoculated Roma tomatoes were studied. Salmonella enterica strains, serotype Montevideo, Javiana and Baildon, were used to experimentally inoculate the food product. After ClO₂ treatments, tomatoes were stored at room temperature for 28 days. Inherent microbial population, change in tomato color, and chlorine dioxide gas residuals were evaluated. ANOVA analysis showed that both ClO₂ concentration and treatment time were significant (p < 0.01) for Salmonella inactivation. Surviving Salmonella populations of 3.09, 2.17 and 1.16 log CFU/cm² were obtained treating tomatoes with 8 mg/l ClO₂ for 60 s, 10 mg/l ClO₂ for 120 s, and 10 mg/l for 180 s, respectively (initial Salmonella population: 6.03 ± 0.11 log CFU/cm²). The selected treatments significantly reduced background microflora (p < 0.05), while fruit color and residual contents were not significantly different (p > 0.05), as compared to the control. Results suggest the potential for high-concentration-short-time treatments ClO₂ gas as an effective pathogen inactivation technology for large-scale produce packing operations.     

Use of marination for controlling Salmonella enterica and Listeria monocytogenes in raw beef

The effect of marination on the survival and growth of the pathogens Salmonella enterica and Listeria monocytogenes on beef pieces was investigated.     

Salmonella Typhimurium internalization is variable in leafy vegetables and fresh herbs

Despite washing and decontamination, outbreaks linked to consumption of fresh or minimally-processed leafy greens have been increasingly reported in recent years. In order to assure the safety of produce it is necessary to gain knowledge regarding the exact routes of contamination. Leaf internalization through stomata was previously reported as a potential route of contamination, which renders food-borne pathogens protected from washing and disinfection by sanitizers. In the present study we have examined the incidence (percentage of microscopic fields harboring ≥ 1 GFP-tagged bacteria) of Salmonella Typhimurium on the surface and underneath the epidermis in detached leaves of seven vegetables and fresh herbs. The incidence of internalized Salmonella varied considerably among the different plants. The highest incidence was observed in iceberg lettuce (81 ± 16%) and arugula leaves (88 ± 16%), while romaine (16 ± 16%) and red-lettuce (20 ± 15%), showed significantly lower incidence (P < 0.05). Internalization incidence in fresh basil was 46 ± 12%, while parsley and tomato leaves demonstrated only marginal internalization (1.9 ± 3.3% and 0.56 ± 1.36%, respectively). Internalization of Salmonella in iceberg lettuce largely varied (0-100%) through a 2 year survey, with a higher incidence occurring mainly in the summer. These results imply that Salmonella internalization occurs in several leafy vegetables and fresh herbs, other than iceberg lettuce, yet the level of internalization largely varies among plants and within the same crop. Since internalized bacteria may evade disinfection, it is of great interest to identify plants which are more susceptible to bacterial internalization, as well as plant and environmental factors that affect internalization.     

Bactericidal effects of nonthermal low-pressure oxygen plasma on S. typhimurium LT2 attached to fresh produce surfaces

This work investigates the feasibility of nonthermal low-pressure oxygen plasma on sanitization of spinach, lettuce, tomato and potato surfaces from Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (Salmonella typhimurium LT2). It was shown that the time of exposure and plasma power density were two critical parameters influencing the bactericidal efficiency. Surface roughness and hydrophobicity did not influence the sanitization of produce. Oxygen plasma was more effective than washing with 3% H₂O₂ on eliminating S. typhimurium LT2 on spinach. Plasma treatment chemically changed a very thin section of tomato wax cuticle layer by oxidation reaction and decomposition of carbon chains, which could readily and completely be removed by water. Overall, this study confirms that nonthermal oxygen plasma can be a new effective method of sanitization for fresh produce.     

Inhibition of polymerase chain reaction for the detection of Escherichia coli O157:H7 and Salmonella enterica on walnut kernels

The aim of this study was to determine whether Escherichia coli O157:H7 can be reliably detected and isolated from walnut kernels using standard methods of analysis. The limit of detection approached 1 cell per analytical unit (25 g) for E. coli O157:H7 on walnut kernels enriched in modified tryptic soy broth with 20 μg/ml novobiocin and plating onto selective agar media. The presence of PCR inhibitors in walnut kernels was indicated by the failure to detect E. coli O157:H7 from culture positive enrichment broths analysed by PCR, with two separate polymerase and reagent compositions (Dupont BAX E. coli O157:H7 MP system, Promega GoTaq Green for stx) and three methods of template preparation (DuPont BAX, Qiagen DNeasy, Bio-Rad InstaGene). PCR inhibition was overcome by 1:100 dilution in TE buffer of the DNeasy or InstaGene template. PCR inhibition was not relieved by dilution of the BAX template. Similar results were observed for walnut kernels inoculated with Salmonella enterica and analysed for invA, indicating that PCR inhibition is not specific to the organism or primer/template. These results indicate that analysis of walnut kernels for pathogens should be with culture based methods or use protocols for DNA template preparation modified to remove or dilute inhibitors and the need for internal amplification controls in PCR methods.     

Inactivation of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves by X-ray

Several recent foodborne disease outbreaks associated with leafy green vegetables, including spinach, have been reported. X-ray is a non-thermal technology that has shown promise for reducing pathogenic and spoilage bacteria on spinach leaves. Inactivation of inoculated Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica and Shigella flexneri on spinach leaves using X-ray at different doses (0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 1.5 and 2.0 kGy) was studied. The effect of X-ray on color quality and microflora counts (mesophilic counts, psychrotrophic counts and yeast and mold counts) of untreated and treated spinach was also determined. A mixture of three strains of each tested organism was spot inoculated (100 μl) onto the surface of spinach leaves (approximately 8–9 log ml⁻¹), separately, and air-dried, followed by treatment with X-ray at 22 °C and 55–60% relative humidity. Surviving bacterial populations on spinach leaves were evaluated using a nonselective medium (tryptic soy agar) with a selective medium overlay for each bacteria; E. coli O157:H7 (CT-SMAC agar), L. monocytogenes (MOA), and S. enterica and S. flexneri (XLD). More than a 5 log CFU reduction/leaf was achieved with 2.0 kGy X-ray for all tested pathogens. Furthermore, treatment with X-ray significantly reduced the initial inherent microflora on spinach leaves and inherent levels were significantly (p < 0.05) lower than the control sample throughout refrigerated storage for 30 days. Treatment with X-ray did not significantly affect the color of spinach leaves, even when the maximum dose (2.0 kGy) was used.     

Prevalence and characterization of Salmonella enterica in dried milk-related infant foods in Shaanxi,China

The aim of this study was to investigate the existence and characteristics of Salmonella enterica in dried milk-related infant foods. Twenty-four (3.4%) of 705 samples, including 5 (2.0%) of 246 powdered infant formula, 18 (4.0%) of 445 infant rice cereal, and 1 (7.1%) of 14 other infant foods, were positive for Salmonella. Fifteen serotypes were identified in 40 Salmonella isolates; Salmonella Duesseldorf (15.0%) and Salmonella Indiana (15.0%) were more frequently detected than other serotypes. Resistance to chloramphenicol (82.5%) was most common, followed by tetracycline (57.5%), ceftiofur (52.5%), kanamycin (52.5%), streptomycin (50.0%), gentamycin (45.0%), nalidixic acid (35.0%), ceftriaxone (32.5%), ciprofloxacin (25.0%), amikacin (20.0%), and cefoxitin (15.0%). Twenty-eight (70.0%) isolates were resistant to ≥8 antimicrobials, with 5 (12.5%) being resistant to 14 antimicrobials. Amino acid substitutions in gyrase A (GyrA) were most frequently detected as Ser83Arg/Asp87Glu and in p53-associated Parkin-like cytoplasmic protein (ParC), they were all Ser80Arg; the quinolone resistance gene qnrS (47.5%) was commonly detected as well as aminoglycoside acetyltransferase [aac(6′)-Ib; 25.0%], qnrA (17.5%), and qnrB (15.0%) genes. Thirty distinct pulsed-field gel electrophoresis patterns were identified among 40 isolates; no identical pulsed-field gel electrophoresis pattern was detected among Salmonella isolates with the same serovar that was recovered in 2010 and 2012. Our results suggest that dried milk-related infant foods could be contaminated with Salmonella and highlight that the dangers to infant health should not be neglected.     

Biofilm formation of Salmonella Typhimurium on stainless steel and acrylic surfaces as affected by temperature and pH level

The effect of temperature (28, 37 and 42 °C) and pH (6 and 7) on the biofilm formation capability of Salmonella Typhimurium on stainless steel and acrylic was investigated. The rate of biofilm formation increased with increasing temperature and pH, while the number of attached cells after 240 h decreased with increasing temperature and was not different between pH 6 and 7. The surface hydrophobicity of bacterial cells was not significantly (p > 0.05) different among tested conditions. Electron-donating/accepting properties changed with pH and temperature, although these changes did not correlate with the ability to form biofilms under respective conditions. Attachment of S. Typhimurium showed a preference for stainless steel compared to acrylic surfaces under all conditions tested. The results suggest that salmonellae were less adherent to acrylic than to stainless steel surfaces; thus, acrylic-type surfaces should be considered for use in the food industry over stainless steel where applicable. The rate of biofilm formation increased at higher temperatures and pH levels within the tested ranges. Hurdle technology using lower temperatures reduced pH may help delay biofilm formation on food contact surfaces contaminated with S. Typhimurium.     

Inhibitory effect of sour orange (Citrus aurantium) juice on Salmonella Typhimurium and Listeria monocytogenes

The sour orange (Citrus aurantium) juice is commonly used as flavoring and acidifying agent for vegetable salads and appetizers in Turkey. It was aimed to determine the survival and growth pattern of Salmonella Typhimurium and Listeria monocytogenes in sour orange juice. Different concentrations of neutralized and un-neutralized juice samples were inoculated with each of the test microorganisms (∼6 log CFU/mL) separately and then incubated at 4 °C and 37 °C for seven days. It was detected both of the test microorganisms could survive and even grow in neutralized juice samples at 37 °C for two days. However, none of them could survive at the end of seventh day of incubation at 37 °C. Low incubation temperature (+4 °C) increased the survival of the tested microorganisms. Also, it was detected that L. monocytogenes were less resistant to the variable conditions than S. Typhimurium. It was concluded that the antimicrobial effect of sour orange juice mainly depends on the low pH value of the product. However, incubation time and temperature are also effective on the survival of the tested pathogens.     

Effect of sample preparation and bacterial concentration on Salmonella enterica detection in poultry meat using culture methods and PCR assaying of preenrichment broths

We evaluated the sensitivity of a PCR assay in the detection of Salmonella enterica at the broth preenrichment step of poultry meat. A total of 162 retail poultry meat samples, which were prepared by manual massaging, stomacher or no homogenization were compared for Salmonella recovery. Using these homogenization methods, the PCR assay at the broth preenrichment step detected Salmonella in, respectively, 48.9%, 62.2% and 50.0% of meat and giblet samples detected as Salmonella-positive using the culture method. In ground chicken, however, Salmonella was detected in 21.7% of samples treated by stomacher homogenization, compared to 40.7% and 48% of untreated and hand-massaged samples, respectively. These results suggest that stomaching of ground chicken causes excessive effusion of food constituents, which affects PCR results. Using the most probable number (MPN) technique, Salmonella was detected at under 1.0 CFU/g in 12 ground chicken samples and under 10³ CFU/ml of broth in seven of the 12 broth-enriched samples, which considered the minimum concentration detectable by PCR assay. These results show that Salmonella detection using routine PCR assays is difficult in poultry meat, and in particular ground chicken, due to low amounts of Salmonella and the presence of inhibitors.     

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R² > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.     

Cold atmospheric gas plasma disinfection of chicken meat and chicken skin contaminated with Listeria innocua

Gas plasmas generated at atmospheric pressure and ambient temperatures offer a possible decontamination method for poultry products. The efficacy of cold atmospheric gas plasmas for decontaminating chicken skin and muscle inoculated with Listeria innocua was examined. Optimization of operating conditions for maximal bacterial inactivation was first achieved using membrane filters on which L. innocua had been deposited. Higher values of AC voltage, excitation frequency and the presence of oxygen in the carrier gas resulted in the greatest inactivation efficiency, and this was confirmed with further studies on chicken muscle and skin. Under optimal conditions, a 10 s treatment gave > 3 log reductions of L. innocua on membrane filters, an 8 min treatment gave 1 log reduction on skin, and a 4 min treatment gave > 3 log reductions on muscle. These results show that the efficacy of gas plasma treatment is greatly affected by surface topography. Scanning electron microscopy (SEM) images of chicken muscle and skin revealed surface features wherein bacteria could effectively be protected from the chemical species generated within the gas plasma. The developments in gas plasma technology necessary for its commercial application to foods are discussed.