Biocontrol of the food-borne pathogens Listeria monocytogenes and Salmonella enterica serovar Poona on fresh-cut apples with naturally occurring bacterial and yeast antagonists

Fresh-cut apples contaminated with either Listeria monocytogenes or Salmonella enterica serovar Poona, using strains implicated in outbreaks, were treated with one of 17 antagonists originally selected for their ability to inhibit fungal postharvest decay on fruit. While most of the antagonists increased the growth of the food-borne pathogens, four of them, including Gluconobacter asaii (T1-D1), a Candida sp. (T4-E4), Discosphaerina fagi (ST1-C9), and Metschnikowia pulcherrima (T1-E2), proved effective in preventing the growth or survival of food-borne human pathogens on fresh-cut apple tissue. The contaminated apple tissue plugs were stored for up to 7 days at two different temperatures. The four antagonists survived or grew on the apple tissue at 10 or 25 degrees C. These four antagonists reduced the Listeria monocytogenes populations and except for the Candida sp. (T4-E4), also reduced the S. enterica serovar Poona populations. The reduction was higher at 25 degrees C than at 10 degrees C, and the growth of the antagonists, as well as pathogens, increased at the higher temperature.     

Biocontrol of Listeria monocytogenes on Fresh-Cut Produce by Treatment with Lytic Bacteriophages and a Bacteriocin

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.     

Autonomous Growth of Isolated Single Listeria monocytogenes and Salmonella enterica Serovar Typhimurium Cells in the Absence of Growth Factors and Intercellular Contact

The aim of this study was to observe growth of isolated single bacterial cells in the absence of growth factors and intercellular contact. In order to exclude stochastic uncertainties induced by dilution series, a new micromanipulation method was developed to ensure explicit results under visual control. This was performed with particular care for production of single prokaryotic cells and subsequent investigation of their autonomous growth. Over 450 single isolated Listeria monocytogenes and Salmonella enterica subsp. enterica serovar Typhimurium cells in lag, log, and stationary growth phases were investigated by this method, which included thoroughly washing the cells. The proportion of living cells within the initial cultures was compared to the proportion of positive samples after enrichment of the separated single cells. This resulted in P values of ≥0.05 using the chi-square test for statistical analysis, indicating no significant difference, and clearly demonstrates reproduction of isolated single bacterial cells without the need for growth factors or intercellular contact. Ease of handling of the apparatus and good performance of the cleaning procedures were achieved, as was validation of the method, demonstrating its suitability for routine laboratory use.The possibility of independent growth of isolated single prokaryotic cells has been discussed recently and remains controversial (15). Undoubtedly, cell-to-cell communication plays a key role in the establishment and development of bacterial communities. Both physical and chemical factors influence the organization of biofilms, sporulation, and resuscitation of bacterial populations. The analogy of the chemical factors to eukaryotic pheromones, as well as their role in bacterial cell division, has been postulated (34). These facts are evident and have been well investigated, and, in summary, the necessity of intercellular contacts, population effects, and intrinsic growth factors such as resuscitating promoting factor (rpf) is generally supposed (24, 36). Nevertheless, when it comes to cell division and growth of low inocula of prokaryotic cells, some questions appear to remain open. Considering evolutionary developments, asexual reproduction is a prerequisite for survival of single organisms in the environment and one of the necessities for the success of the prokaryotic kingdom. Taken the other way, asexual reproduction also suggests the possibility of independent growth of isolated single bacterial cells. The scientific community remains divided over this possibility, not least because of the possible heterogeneity of bacterial cultures in terms of the physiological status of every single cell and the effect of a Poisson distribution in highly diluted cultures, which influence experimental setups and results (17).If one viable prokaryotic cell is to be shown to be capable of generating a population of daughter cells, a prerequisite is its isolation and physical manipulation as an individual cell. Simple serial dilution protocols, such as the most probable number (MPN) method, achieve this task, but they suffer from a lack of certainty that the diluted solution indeed contains only one viable cell, free from any adherent growth factors. In such cases the accuracy of the data obtained by multiple dilution procedures becomes uncertain regarding the Poisson distribution of highly diluted bacterial cell (≤10 CFU/ml) suspensions (11, 32).Combined microinjection and micromanipulation methods are alternative applications. These are widespread routine techniques that have been developed recently for large eukaryotic cells (14, 38). However, application of these methods to prokaryotic cells leads to new physical conditions defined by the technical equipment secondary to the size of the handled cells (≤1 μm). These include limitations of fluorescence microscopy, general microscopic analysis, and photographic documentation. Over the past 30 years several attempts to improve the management of single prokaryotic cells using micromanipulation techniques have been made (10). The core issue of these studies has been isolation of single microbial cells from mixed populations under direct visual control (9, 13).In contrast, the aim of this study was to develop a method capable of serial manipulation of single prokaryotic cells under visual control. This was accomplished to produce series of single prokaryotic cells and subsequently to investigate their autonomous growth. The growth of single Listeria monocytogenes and Salmonella enterica subspecies enterica serovar Typhimurium cells was investigated in highly diluted buffer systems without facultative growth factors being transferred from the primary enrichment. In this study over 450 single cells isolated from different growth phases (lag phase, log phase, and stationary phase) were manipulated using a novel single bacterial cell manipulation (SBCM) technique.     

Pectin and Xyloglucan Influence the Attachment of Salmonella enterica and Listeria monocytogenes to Bacterial Cellulose-Derived Plant Cell Wall Models

Minimally processed fresh produce has been implicated as a major source of foodborne microbial pathogens globally. These pathogens must attach to the produce in order to be transmitted. Cut surfaces of produce that expose cell walls are particularly vulnerable. Little is known about the roles that different structural components (cellulose, pectin, and xyloglucan) of plant cell walls play in the attachment of foodborne bacterial pathogens. Using bacterial cellulose-derived plant cell wall models, we showed that the presence of pectin alone or xyloglucan alone affected the attachment of three Salmonella enterica strains (Salmonella enterica subsp. enterica serovar Enteritidis ATCC 13076, Salmonella enterica subsp. enterica serovar Typhimurium ATCC 14028, and Salmonella enterica subsp. indica M4) and Listeria monocytogenes ATCC 7644. In addition, we showed that this effect was modulated in the presence of both polysaccharides. Assays using pairwise combinations of S. Typhimurium ATCC 14028 and L. monocytogenes ATCC 7644 showed that bacterial attachment to all plant cell wall models was dependent on the characteristics of the individual bacterial strains and was not directly proportional to the initial concentration of the bacterial inoculum. This work showed that bacterial attachment was not determined directly by the plant cell wall model or bacterial physicochemical properties. We suggest that attachment of the Salmonella strains may be influenced by the effects of these polysaccharides on physical and structural properties of the plant cell wall model. Our findings improve the understanding of how Salmonella enterica and Listeria monocytogenes attach to plant cell walls, which may facilitate the development of better ways to prevent the attachment of these pathogens to such surfaces.     

Antimicrobial Effects of Mustard Flour and Acetic Acid against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10⁶ to 10⁷ CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Correlation of Phenotype with the Genotype of Egg-Contaminating Salmonella enterica Serovar Enteritidis

The genotype of Salmonella enterica serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whereas the PT4 genome to which they were compared, as well as a PT4 field isolate, had a DNA sequence with some similarity to the bacteriophage ST64b sequence. Phenotype MicroArray analysis indicated that the two PT13A strains and the PT4 field isolate had similar respiratory activity profiles at 37°C. However, the wild-type S. enterica serovar Enteritidis PT13A strain grew significantly better in 20% more of the 1,920 conditions tested when it was assayed at 25°C than the biofilm-forming PT13A strain grew. Statistical analysis of the respiratory activity suggested that S. enterica serovar Enteritidis PT4 had a temperature-influenced dimorphic metabolism which at 25°C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37°C the metabolism was nearly identical to that of the wild-type PT13A strain. Although it is possible that lysogenic bacteriophage alter the balance of phage types on a farm either by lytic competition or by altering the metabolic processes of the host cell in subtle ways, the different physiologies of the S. enterica serovar Enteritidis strains correlated most closely with minor, rather than major, genomic changes. These results strongly suggest that the pandemic of egg-associated human salmonellosis that came into prominence in the 1980s is primarily an example of bacterial adaptive radiation that affects the safety of the food supply.     

Growth of Listeria monocytogenes and Yersinia enterocolitica on Cooked Modified-Atmosphere-Packaged Poultry in the Presence and Absence of a Naturally Occurring Microbiota

Cooked poultry cuts were inoculated with five-strain composite mixtures of either Listeria monocytogenes or Yersinia enterocolitica (1,000 CFU/150-g piece), packaged in 44:56 CO₂-N₂, and stored at 3.5, 6.5, or 10°C for up to 5 weeks. Both L. monocytogenes and Y. enterocolitica grew under all test conditions. The presence of a naturally occurring microbiota did not influence the growth of either pathogen. Addition of lactate with the shelf life extender ALTA 2341 lengthened the lag phases of L. monocytogenes and Y. enterocolitica but did not prevent their growth.     

Salmonella enterica Serovar Typhimurium and Listeria monocytogenes Acid Tolerance Response Induced by Organic Acids at 20°C: Optimization and Modeling

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20°C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (t_s), the log-linear decline (k_max), and the magnitude of a critical component (C). The magnitude of k_max gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.     

Evaluation of Near-Infrared Pasteurization in Controlling Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Ready-To-Eat Sliced Ham

This study was conducted to investigate the efficacy of near-infrared (NIR) heating to reduce Salmonella enterica serovar Typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes in ready-to-eat (RTE) sliced ham compared to conventional convective heating, and the effect of NIR heating on quality was determined by measuring the color and texture change. A cocktail of three pathogens was inoculated on the exposed or protected surfaces of ham slices, followed by NIR or conventional heating at 1.8 kW. NIR heating for 50 s achieved 4.1-, 4.19-, and 3.38-log reductions in surface-inoculated S. Typhimurium, E. coli O157:H7, and L. monocytogenes, respectively, whereas convective heating needed 180 s to attain comparable reductions for each pathogen. There were no statistically significant (P > 0.05) differences in reduction between surface- and internally inoculated pathogens at the end of NIR treatment (50 s). However, when treated with conventional convective heating, significant (P < 0.05) differences were observed at the final stages of the treatment (150 and 180 s). Color values and texture parameters of NIR-treated (50-s treatment) ham slices were not significantly (P > 0.05) different from those of nontreated samples. These results suggest that NIR heating can be applied to control internalized pathogens as well as surface-adhering pathogens in RTE sliced meats without affecting product quality.     

Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10 degrees C but increased significantly on fresh-cut honeydew melons stored at 10 degrees C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.     

Survival of Salmonella enteritidis and Listeria monocytogenes on salad vegetables

The influence of initial head-spaces of air – 4.9% CO₂/2.1% O₂/93% N₂ and 5% CO₂/5.2% O₂/89.8% N₂ – on Salmonella enteritidis and Listeria monocytogenes, and on microbial association with shredded carrots and lettuce was studied at 4 °C. Both these pathogens survived but did not grow in any vegetable regardless of the packaging system used. Total viable count, lactic acid bacteria and pseudomonads were also monitored. Lactic acid bacteria were the predominant organisms in all samples. The pH dropped significantly during the storage of vegetables.     

Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium,and Listeria monocytogenes in Buffered Peptone Water and Apple Juice

The effect of electric field-induced ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P < 0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P < 0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating.     

Risk Factors Associated with Salmonella and Listeria monocytogenes Contamination of Produce Fields

Identification of management practices associated with preharvest pathogen contamination of produce fields is crucial to the development of effective Good Agricultural Practices (GAPs). A cross-sectional study was conducted to (i) determine management practices associated with a Salmonella- or Listeria monocytogenes-positive field and (ii) quantify the frequency of these pathogens in irrigation and nonirrigation water sources. Over 5 weeks, 21 produce farms in New York State were visited. Field-level management practices were recorded for 263 fields, and 600 environmental samples (soil, drag swab, and water) were collected and analyzed for Salmonella and L. monocytogenes. Management practices were evaluated for their association with the presence of a pathogen-positive field. Salmonella and L. monocytogenes were detected in 6.1% and 17.5% of fields (n = 263) and 11% and 30% of water samples (n = 74), respectively. The majority of pathogen-positive water samples were from nonirrigation surface water sources. Multivariate analysis showed that manure application within a year increased the odds of a Salmonella-positive field (odds ratio [OR], 16.7), while the presence of a buffer zone had a protective effect (OR, 0.1). Irrigation (within 3 days of sample collection) (OR, 6.0), reported wildlife observation (within 3 days of sample collection) (OR, 6.1), and soil cultivation (within 7 days of sample collection) (OR, 2.9) all increased the likelihood of an L. monocytogenes-positive field. Our findings provide new data that will assist growers with science-based evaluation of their current GAPs and implementation of preventive controls that reduce the risk of preharvest contamination.     

Colonization of barley (Hordeum vulgare) with Salmonella enterica and Listeria spp

Colonization of barley plants by the food-borne pathogens Salmonella enterica serovar typhimurium and three Listeria spp. (L. monocytogenes, L. ivanovii, L. innocua) was investigated in a monoxenic system. Herbaspirillum sp. N3 was used as a positive control and Escherichia coli HB101 as a negative control for endophytic root colonization. Colonization of the plants was tested 1-4 weeks after inoculation by determination of CFU, specific PCR assays and fluorescence in situ hybridization (FISH) with fluorescently labelled oligonucleotide probes in combination with confocal laser scanning microscopy (CLSM). Both S. enterica strains were found as endophytic colonizers of barley roots and reached up to 2.3 x 10(6) CFU per g root fresh weight after surface sterilization. The three Listeria strains had 10-fold fewer cell numbers after surface sterilization on the roots and therefore were similar to the results of nonendophytic colonizers, such as E. coli HB101. The FISH/CSLM approach demonstrated not only high-density colonization of the root hairs and the root surface by S. enterica but also a spreading to subjacent rhizodermis layers and the inner root cortex. By contrast, the inoculated Listeria spp. colonized the root hair zone but did not colonize other parts of the root surface. Endophytic colonization of Listeria spp. was not observed. Finally, a systemic spreading of S. enterica to the plant shoot (stems and leaves) was demonstrated using a specific PCR analysis and plate count technique.