Publications by authors named "Teresa Bergholz"

37 Publications

Impact of Chlorinated Water on Pathogen Inactivation during Wheat Tempering and Resulting Flour Quality.

J Food Prot 2022 08;85(8):1210-1220

Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824.

Abstract: Outbreaks of enteric pathogens linked to wheat flour have led the wheat milling industry to seek solutions addressing this food safety concern. Chlorinated water at 400 to 700 ppm has been used in the flour milling industry as a tempering aid to control growth of yeast and mold in tempering bins. However, the effectiveness of chlorinated water for inactivating enteric pathogens on wheat kernels was unknown. Five strains of Shiga toxin-producing Escherichia coli and two strains of Salmonella were inoculated onto hard red spring wheat at 7 log CFU/g and stored at room temperature for 1 month. Inoculated wheat was tempered with four concentrations (0, 400, 800, and 1,200 ppm) of chlorinated water (pH 6.5). The reduction due to chlorine was determined by calculating change in microbial loads at each chlorine level by using the response at 0 ppm as a reference. Uninoculated wheat tempered with chlorinated water was used to measure flour quality parameters. Changes in pathogen population over 18 h ranged from -2.35 to -0.30 log CFU/g with 800 ppm of chlorinated water and were not significantly different from changes at 400 and 1,200 ppm. Significant (P < 0.05) differences in the extent of reduction were observed among strains. However, the effect of chlorinated water at reducing native microbes on wheat kernels was minimal, with an average reduction of 0.39 log CFU/g for all concentrations. No significant (P > 0.05) changes occurred in flour quality and gluten functionality or during bread making for grains tempered at 400 and 800 ppm of chlorinated water. There were small but significant (P < 0.05) changes in flour protein content, final viscosity, and water absorption when tempered with 1,200 ppm of chlorinated water. The data showed that the level of chlorinated water currently used in industry for tempering could reduce enteric pathogen numbers by 1.22 log CFU/g for Shiga toxin-producing Escherichia coli and 2.29 log CFU/g for Salmonella, with no significant effects on flour quality and gluten functionality.

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http://dx.doi.org/10.4315/JFP-22-076DOI Listing
August 2022

Fate of Salmonella and Enterohemorrhagic Escherichia coli on Wheat Grain.

J Food Prot 2021 12;84(12):2109-2115

Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824, USA.

Abstract: Wheat flour has been connected to outbreaks of foodborne illnesses with increased frequency in recent years, specifically, outbreaks involving Salmonella enterica and enterohemorrhagic Escherichia coli (EHEC). However, there is little information regarding the survival of these pathogens on wheat grain during long-term storage in a low-moisture environment. This study aims to evaluate the long-term survival of these enteric pathogens on wheat grain over the course of a year. Hard red spring wheat was inoculated with strains of four serovars of Salmonella (Enteritidis, Agona, Tennessee, and Montevideo) and six serotypes of EHEC (O157:H7, O26:H11, O121:H19, O45:NM, O111:H8, and O103:H2) in triplicate, sealed in Mylar bags to maintain the water activity, and stored at room temperature (22 ± 1°C). The survival of each pathogen was evaluated by plating onto differential media. Viable counts of strains from all four serovars of Salmonella (Enteritidis, Agona, Tennessee, and Montevideo) were detected on wheat grain stored at room temperature (22 ± 1°C) for the duration of the study (52 weeks). Viable counts of strains from EHEC serotypes O45:NM, O111:H8, and O26:H11 were only detected for 44 weeks, and strains from serotypes O157:H7, O121:H19, and O103:H2 were only detected for 40 weeks until they passed below the limit of detection (2.0 log CFU/g). The D-values were found to be significantly different between Salmonella and EHEC (adjusted P ≤ 0.05) with Salmonella D-values ranging from 22.9 ± 2.2 weeks to 25.2 ± 1.0 weeks and EHEC D-values ranging from 11.4 ± 0.6 weeks to 13.1 ± 1.8 weeks. There were no significant differences among the four Salmonella strains or among the six EHEC strains (adjusted P > 0.05). These observations highlight the wide range of survival capabilities of enteric pathogens in a low-moisture environment and confirm these pathogens are a food safety concern when considering the long shelf life of wheat grain and its products.

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http://dx.doi.org/10.4315/JFP-21-076DOI Listing
December 2021

Survival and thermal resistance among four Salmonella serovars inoculated onto flaxseeds.

Food Microbiol 2020 Oct 23;91:103516. Epub 2020 Apr 23.

Department of Microbiological Sciences North Dakota State University, Fargo, ND, 58102, USA. Electronic address:

Thermal resistance among Salmonella serovars has been shown to vary, however, such data are minimal for Salmonella inoculated onto low moisture foods. We evaluated survival and subsequent thermal resistance for 32 strains of Salmonella from four serovars (Agona, Enteritidis, Montevideo, and Tennessee) on flaxseed over 24 weeks. After inoculation, flaxseeds were adjusted to a = 0.5 and stored at 22 °C. After 24 weeks at 22 °C, strains of serovar Agona had a significantly slower rate of reduction compared to those of Enteritidis and Montevideo (adj. p < 0.05). Inoculated flaxseeds were processed at 71 °C with vacuum steam pasteurization at 4 time points during storage. Average initial D values ranging from 1.0 to 1.5 min were similar across serovars. Over 24 weeks, D varied in a serovar-dependent manner. D at 8, 16, and 24 weeks did not change significantly for Enteritidis and Montevideo but did for Tennessee and Agona. While significant, the differences in D over time were less than 1 min, indicating that storage time prior to heat treatment would have a minimal effect on the processing time required to inactivate Salmonella on flaxseed.
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http://dx.doi.org/10.1016/j.fm.2020.103516DOI Listing
October 2020

Effect of Vacuum Steam Treatment of Hard Red Spring Wheat on Flour Quality and Reduction of Escherichia coli O121 and Salmonella Enteritidis PT 30.

J Food Prot 2020 May;83(5):836-843

Department of Plant Sciences (ORCID: https://orcid.org/0000-0003-0238-5947 [S.S.]) and.

Abstract: Recent outbreaks traced to contaminated flour have created a need in the milling industry for a process that reduces pathogens in wheat while maintaining its functional properties. Vacuum steam treatment is a promising technology for treatment of low-moisture foods. Traditional thermal treatment methods can compromise wheat functionality due to high temperatures; thus, maintaining the functional quality of the wheat protein was critical for this research. The objective of this study was to evaluate the effect of vacuum steam treatment of hard red spring (HRS) wheat kernels on final flour quality and the overall efficacy of vacuum stream treatment for reducing pathogens on HRS wheat kernels. HRS wheat samples were treated with steam under vacuum at 65, 70, 75, and 85°C for 4 and 8 min. Significant changes in dough and baked product functionality were observed for treatments at ≥70°C. Treatment time had no significant effect on the qualities evaluated. After determining that vacuum steam treatment at 65°C best preserved product quality, HRS wheat was inoculated with Escherichia coli O121 and Salmonella Enteritidis PT 30 and processed at 65°C for 0, 2, 4, 6, or 8 min. The treatments achieved a maximum average reduction of 3.57 ± 0.33 log CFU/g for E. coli O121 and 3.21 ± 0.27 log CFU/g for Salmonella. Vacuum steam treatment could be an effective pathogen inactivation method for the flour milling industry.

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http://dx.doi.org/10.4315/JFP-19-491DOI Listing
May 2020

Pre-Harvest Survival and Post-Harvest Chlorine Tolerance of Enterohemorrhagic on Lettuce.

Toxins (Basel) 2019 11 19;11(11). Epub 2019 Nov 19.

Department of Microbiological Sciences, North Dakota State University, Fargo, ND 58102, USA.

In the field, foodborne pathogens such as enterohemorrhagic (EHEC) are capable of surviving on produce over time, yet little is known about how these pathogens adapt to this environment. To assess the impact of pre-harvest environmental conditions on EHEC survival, we quantified survival on romaine lettuce under two relative humidity (75% and 45%) and seasonal conditions (March and June). Greenhouse-grown lettuce was spray-inoculated with EHEC and placed in a growth chamber, mimicking conditions typical for June and March in Salinas Valley, California. Bacteria were enumerated on days 0, 1, 3, and 5 post-inoculation. Overall, we found that the effect of relative humidity on EHEC survival depended on the seasonal conditions. Under June seasonal conditions, higher relative humidity led to lower survival, and lower relative humidity led to greater survival, five days post-inoculation. Under March seasonal conditions, the impact of relative humidity on EHEC survival was minimal over the five days. The bacteria were also tested for their ability to survive a chlorine decontamination wash. Inoculated lettuce was incubated under the June 75% relative humidity conditions and then washed with a 50 ppm sodium hypochlorite solution (40 ppm free chlorine). When incubated under June seasonal conditions for three to five days, EHEC strains showed increased tolerance to chlorine (adj. < 0.05) compared to chlorine tolerance upon inoculation onto lettuce. This indicated that longer incubation on lettuce led to greater EHEC survival upon exposure to chlorine. Subsequent transcriptome analysis identified the upregulation of osmotic and oxidative stress response genes by EHEC after three and five days of incubation on pre-harvest lettuce. Assessing the physiological changes in EHEC that occur during association with pre-harvest lettuce is important for understanding how changing tolerance to post-harvest control measures may occur.
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http://dx.doi.org/10.3390/toxins11110675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891304PMC
November 2019

Evidence of hypervirulence in clonal complex 14.

J Med Microbiol 2019 Nov;68(11):1677-1685

Department of Microbiological Sciences, North Dakota State University, Fargo, ND 58108, USA.

is a foodborne pathogen that causes central nervous system (CNS) and maternal-neonatal (MN) infections, bacteremia (BAC), and gastroenteritis in humans and ruminants. Specific clonal complexes (CC) have been associated with severe listeriosis cases, however, less is known about differences among subgroup virulence patterns. This study aimed to assess variation in virulence across different CC and clinical outcomes. larvae were used to compare virulence phenotypes of 34 . strains representing isolates from CC1, CC6 (from lineage I), and CC7, CC9, CC14, CC37 and CC204 (from lineage II) classified by clinical outcome: BAC, CNS and MN infection. Larvae survival, LD, cytotoxicity, health index scores and bacterial concentrations post-infection were evaluated as quantifiable indicators of virulence. Isolates belonging to CC14 and MN-associated infections are hypervirulent in as they led to lower survival rates and health index scores, as well as reduced cytotoxic effects when compared to other CC and clinical outcomes included here. CC14 isolates also showed increased bacterial concentrations at 8 and 24 h post-infection, indicating ability to survive the initial immune response and proliferate within larvae. Subgroups of possess different virulence phenotypes that may be associated with niche-specificity. While hypervirulent clones have been identified so far in lineage I, our data demonstrate that hypervirulent clones are not restricted to lineage I, as CC14 belongs to lineage II. Identification of subgroups with a higher ability to cause disease may facilitate surveillance and management of listeriosis.
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http://dx.doi.org/10.1099/jmm.0.001076DOI Listing
November 2019

in Soils Amended with Heat-Treated Poultry Pellets Survived Longer than Bacteria in Unamended Soils and More Readily Transferred to and Persisted on Spinach.

Appl Environ Microbiol 2019 05 2;85(10). Epub 2019 May 2.

U.S. Department of Agriculture, Agricultural Research Service, Northeast Area, Environmental Microbial Food Safety Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland, USA

Untreated biological soil amendments of animal origin (BSAAO) are commonly used as biological fertilizers but can harbor foodborne pathogens like , leading to potential transfer from soils to fruits and vegetables intended for human consumption. Heat-treated poultry pellets (HTPP) can provide produce growers with a slow-release fertilizer with a minimized risk of pathogen contamination. Little is known about the impact of HTPP-amended soil on the survival of The contributions of RpoS and formation of viable but nonculturable cells to survival in soils are also inadequately understood. We quantified the survival of subsp. serovar Newport wild-type (WT) and -deficient (Δ mutant) strains in HTPP-amended and unamended soil with or without spinach plants over 91 days using culture and quantitative PCR methods with propidium monoazide (PMA-qPCR). Simulated "splash" transfer of Newport from soil to spinach was evaluated at 35 and 63 days postinoculation (dpi). The Newport WT and Δ mutant reached the limit of detection, 1.0 log CFU/g (dry weight), in unamended soil after 35 days, whereas 2 to 4 log CFU/g (dry weight) was observed for both WT and Δ mutant strains at 91 dpi in HTPP-amended soil. Newport levels in soils determined by PMA-qPCR and plate count methods were similar ( > 0.05). HTPP-amended soils supported higher levels of Newport transfer to and survival on spinach leaves for longer periods of time than did unamended soils ( < 0.05). Newport introduced to HTPP-amended soils survived for longer periods and was more likely to transfer to and persist on spinach plants than was Newport introduced to unamended soils. Heat-treated poultry pellets (HTPP) often are used by fruit and vegetable growers as a slow-release fertilizer. However, contamination of soil on farms may occur through contaminated irrigation water or scat from wild animals. Here, we show that the presence of HTPP in soil led to increased Newport survival in soil and to greater likelihood of its transfer to and survival on spinach plants. There were no significant differences in survival durations of WT and Δ mutant isolates of Newport. The statistically similar populations recovered by plate count and estimated by PMA-qPCR for both strains in the amended and unamended soils in this study indicate that all viable populations of Newport in soils were culturable.
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http://dx.doi.org/10.1128/AEM.00334-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6498161PMC
May 2019

Survival and Growth of Wild-Type and rpoS-Deficient Salmonella Newport Strains in Soil Extracts Prepared with Heat-Treated Poultry Pellets.

J Food Prot 2019 Mar;82(3):501-506

2 U.S. Department of Agriculture, Agricultural Research Service, Northeast Area, Beltsville Agricultural Research Center, Environmental Microbial Food Safety Laboratory, Beltsville, Maryland 20705 (ORCID: http://orcid.org/000-0002-8585-0308 [M.S.]).

Manure runoff can transfer pathogens to farmlands or to water sources, leading to subsequent contamination of produce. Untreated biological soil amendments, like manure, can be contaminated with foodborne pathogens, such as Salmonella Newport, which may lead to transfer of the pathogen to fruits or vegetables. Studies have reported the occurrence and survival of Salmonella in manure or manure slurries. However, data on the survival and growth of Salmonella Newport is lacking in matrices simulating runoff. We quantified the survival and growth of wild-type (WT) Salmonella Newport and rpoS-deficient (Δ rpoS) strains in sterile and nonsterile soil extracts prepared with (amended) or without (unamended) heat-treated poultry pellets at 25°C. Salmonella Newport WT and Δ rpoS populations reached a maximum cell density of 6 to 8 log CFU/mL in 24 to 30 h in amended and unamended soil extracts and remained in stationary phase for up to 4 days. Salmonella Newport in amended soil extracts exhibited a decreased lag phase (λ , 2.87 ± 1.01 h) and greater maximum cell densities ( N, 6.84 ± 1.25 CFU/mL) compared with λ (20.10 ± 9.53 h) and N (5.22 ± 0.82 CFU/mL) in unamended soil extracts. In amended soil extract, the Δ rpoS strain had no measurable λ , similar growth rates (μ) compared with WT, and a lower N compared with the WT strain. Unamended, nonsterile soil extracts did not support the growth of Salmonella Newport WT and led to a decline in populations for the Δ rpoS strain. Salmonella Newport had lower cell densities in nonsterile soil extracts (5.94 ± 0.95 CFU/mL) than it did in sterile soil extracts (6.66 ± 1.50 CFU/mL), potentially indicating competition for nutrients between indigenous microbes and Salmonella Newport. The most favorable growth conditions were provided by amended sterile and nonsterile soil extracts, followed by sterile, unamended soil extracts for both Salmonella Newport strains. Salmonella Newport may grow to greater densities in amended extracts, providing a route for increased Salmonella levels in the growing environments of produce.
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http://dx.doi.org/10.4315/0362-028X.JFP-18-465DOI Listing
March 2019

Isolation of Bacterial RNA from Foods Inoculated with Pathogens.

Methods Mol Biol 2019 ;1918:129-137

Department of Microbiological Sciences, North Dakota State University, Fargo, ND, USA.

One strategy bacteria use to acclimate to changing environmental conditions is modulation of gene expression. Alterations in gene expression are indicative of activation or repression of certain physiological responses. In order to understand which genetic responses are utilized to cope with various environmental conditions by analyzing transcriptomes, obtaining RNA of high quality, yield, and integrity is paramount. Here, we describe an acid phenol-chloroform method employed to extract RNA from laboratory grown cell cultures, as well as cultures inoculated onto complex matrices such as lettuce and cold-smoked salmon. The method results in high-quality RNA, which can be used for various downstream processes such as cDNA library construction, RNA sequencing, real-time quantitative PCR, and northern analysis. Extraction of RNA from bacterial foodborne pathogens in conjunction with transcriptome sequencing is a useful technique to elucidate pathogens' transcriptional responses.
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http://dx.doi.org/10.1007/978-1-4939-9000-9_10DOI Listing
June 2019

Genomic and phenotypic diversity of Listeria monocytogenes clonal complexes associated with human listeriosis.

Appl Microbiol Biotechnol 2018 Apr 2;102(8):3475-3485. Epub 2018 Mar 2.

Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, MD, 20708, USA.

Listeria monocytogenes is a pathogen of significant concern in many ready to eat foods due to its ability to survive and multiply even under significant environmental stresses. Listeriosis in humans is a concern, especially to high-risk populations such as those who are immunocompromised or pregnant, due to the high rates of morbidity and mortality. Whole genome sequencing has become a routine part of assessing L. monocytogenes isolated from patients, and the frequency of different genetic subtypes associated with listeriosis is now being reported. The recent abundance of genome sequences for L. monocytogenes has provided a wealth of information regarding the variation in core and accessory genomic elements. Newly described accessory genomic regions have been linked to greater virulence capabilities as well as greater resistance to environmental stressors such as sanitizers commonly used in food processing facilities. This review will provide a summary of our current understanding of stress response and virulence phenotypes of L. monocytogenes, within the context of the genetic diversity of the pathogen.
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http://dx.doi.org/10.1007/s00253-018-8852-5DOI Listing
April 2018

Microbial and Chemical Shelf-Life of Vacuum Steam-Pasteurized Whole Flaxseed and Milled Flaxseed.

J Food Sci 2018 Feb 19;83(2):300-308. Epub 2018 Jan 19.

the Dept. of Plant Sciences, North Dakota State Univ., Fargo, ND 58108, U.S.A.

Flaxseed is an oilseed with many health benefits. Flaxseed may be consumed raw or in processed form. In the raw form, there is a potential for microbial contamination. Several pasteurization methods have been used to reduce microbial contamination. However, such treatments may affect chemical properties of foods. In this study, vacuum steam-pasteurization was conducted on whole flaxseed and milled flaxseed using 4 different conditions (3 min at 75 °C, 3 min at 90 °C, 9 min at 90 °C, and 3 min at 105 °C). Microbial and chemical shelf-life was monitored for 28 wk (36 wk for aerobic plate counts). Significant reduction (P < 0.05) in microbial counts (total aerobic plate counts, and yeast and mold counts) occurred after pasteurization and during storage of both whole flaxseed and milled flaxseed. Although both the moisture content and a increased after pasteurization, they were similar to the unpasteurized samples during storage. Peroxide value, free fatty acid, headspace volatiles, fatty acid profiles, oil content, and secoisolariciresinol diglucoside (SDG) content were chemical indices measured. Only small changes were observed in the chemical indices after vacuum steam-pasteurization for both pasteurized whole flaxseed and milled flaxseed as compared to the unpasteurized flaxseed at most instances. Vacuum steam-pasteurization can be used as a safe alternative for the microbial reduction of low-moisture products, such as flaxseed, without significantly affecting chemical stability.

Practical Application: Vacuum steam-pasteurization can be effectively used for the treatment of whole flaxseed and milled flaxseed to reduce spoilage microorganisms, such as total aerobes and yeasts and molds. In addition, this pasteurization method had minimal effects on several chemical shelf-life parameters with positive impact on SDG of the processed flaxseed.
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http://dx.doi.org/10.1111/1750-3841.14050DOI Listing
February 2018

Genetic characterization of Listeria monocytogenes from ruminant listeriosis from different geographical regions in the U.S.

Vet Microbiol 2018 Feb 26;215:93-97. Epub 2017 Dec 26.

Department of Microbiological Sciences, North Dakota State University, Fargo, ND 58108, United States. Electronic address:

Listeria monocytogenes infections are an important disease of ruminants worldwide, causing encephalitis, septicemia, and abortions. Ruminant listeriosis can also pose a food safety risk due to the potential for L. monocytogenes to enter the food supply via the farm environment. Data on the genetic diversity of L. monocytogenes from ruminant clinical cases in the United States is limited. Our goal was to assess the genetic diversity of clinical listeriosis isolates from ruminants in the Upper Great Plains states, a population not well-studied, and compare this population to isolates from ruminants in New York State. Multi-locus sequence typing (MLST) was used to classify and compare the genetic diversity of the isolates from the two regions. Loci sequences were compared to all known sequence types using the Pasteur Institute L. monocytogenes MLST database. Four novel sequence types (ST) were identified among the Upper Great Plains isolates, and four new STs were classified in the New York collection. Five STs were found to be common across the 2 geographical regions; ST 1, 7, 191, and 204. Strains of ST 7 were most frequently isolated (7/46 isolates). Strains of ST 91 were all associated with fetal infections from the Upper Great Plains. Our results demonstrate that while there are some subtypes commonly found between the two geographic regions, there are also subtypes distinct to each region.
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http://dx.doi.org/10.1016/j.vetmic.2017.12.021DOI Listing
February 2018

Efficacy of vacuum steam pasteurization for inactivation of Salmonella PT 30, Escherichia coli O157:H7 and Enterococcus faecium on low moisture foods.

Int J Food Microbiol 2017 Mar 6;244:111-118. Epub 2017 Jan 6.

Department of Microbiological Sciences, North Dakota State University, Fargo, ND 58108, United States. Electronic address:

Low moisture foods such as nuts, spices, and seeds have been implicated in several outbreaks due to Salmonella or E. coli O157:H7 contamination. Such foods may be consumed raw, and can be used as ingredients in other food products. While numerous thermal inactivation studies have been conducted for Salmonella on nuts, studies on other seeds and grains are minimal. Product water activity can influence the thermal resistance of pathogens, where thermal resistance increases as water activity decreases, leading to a requirement for higher temperatures and longer exposure times to achieve significant reduction of pathogen numbers. Vacuum steam pasteurization uses steam under vacuum, which can be operated at temperatures above and below 100°C. The objective of this study was to determine the efficacy of vacuum steam pasteurization for inactivation of pathogens on whole flaxseed, quinoa, sunflower kernels, milled flaxseed and whole black peppercorns. The use of E. faecium as a potential surrogate for Salmonella and E. coli O157:H7 in vacuum steam pasteurization was also evaluated. Pasteurization for 1min at 75°C yielded average log reductions of 5.48±1.22, 5.71±0.40 and 5.23±0.61 on flaxseed, 4.29±0.92, 5.89±0.26 and 2.39±0.83 on quinoa, and 4.01±0.74, 5.40±0.83 and 2.99±0.92 on sunflower kernels for Salmonella PT 30, E. coli O157:H7 and E. faecium, respectively. Similarly, on milled flaxseed and black peppercorns average log reductions of 3.02±0.79 and 6.10±0.64CFU/g were observed for Salmonella PT 30 after 1min of treatment at 75°C but, on average, >6.0 log reductions were observed after pasteurization at 85°C. Our data demonstrate that vacuum steam pasteurization can be effectively used to reduce pathogens on these low moisture foods at temperature as low as 75 and 85°C, and that E. faecium may be used as a potential surrogate for Salmonella PT 30 and E. coli O157:H7.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2017.01.003DOI Listing
March 2017

Application of a Nonlinear Model to Transcript Levels of Upregulated Stress Response Gene ibpA in Stationary-Phase Salmonella enterica Subjected to Sublethal Heat Stress.

J Food Prot 2016 07;79(7):1089-96

Department of Biosystems and Agricultural Engineering, Department of Food Science and Human Nutrition, Michigan State University, East Lansing, Michigan 48824, USA.

Sublethal heating, which can occur during slow cooking of meat products, is known to induce increased thermal resistance in Salmonella. However, very few studies have addressed the kinetics of this response. Although several recent studies have reported improved thermal inactivation models that include the effect of prior sublethal history on subsequent thermal resistance, none of these models were based on cellular-level responses to sublethal thermal stress. The goal of this study was to determine whether a nonlinear model could accurately portray the response of Salmonella to heat stress induced by prolonged exposure to sublethal temperatures. To accomplish this, stationary-phase Salmonella Montevideo cultures were subjected to various heating profiles (held at either 40 or 45°C for 0, 5, 10, 15, 30, 60, 90, 180, or 240 min) using a PCR thermal cycler. Differential plating on selective and nonselective media was used to confirm the presence of cellular injury. Reverse transcription quantitative PCR was used to screen the transcript levels of six heat stress-related genes to find candidate genes for nonlinear modeling. Injury was detected in populations of Salmonella held at 45°C for 30, 60, and 90 min and at 40°C for 0, 5, and 90 min (P < 0.05), whereas no significant injury was found at 180 and 240 min (P > 0.05). The transcript levels of ibpA, which codes for a small heat shock protein associated with the ClpB and DnaK-DnaJ-GrpE chaperone systems, showed the greatest increase relative to the transcript levels at 0 min, which was significant at 5, 10, 15, 30, 60, 90, and 180 min at 45°C and at 5, 10, 15, 30, 60, and 90 min at 40°C (P < 0.05). Using ibpA transcript levels as an indicator of adaptation to thermal stress, a nonlinear model for sublethal injury is proposed. The use of variables indicating the physiological state of the pathogen during stress has the potential to increase the accuracy of thermal inactivation models that must account for prolonged exposure to sublethal temperatures.
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http://dx.doi.org/10.4315/0362-028X.JFP-15-377DOI Listing
July 2016

Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing.

Appl Environ Microbiol 2016 02 20;82(3):928-38. Epub 2015 Nov 20.

Centers for Disease Control and Prevention, Atlanta, Georgia, USA

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or "epidemic clones" that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in L. monocytogenes and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.
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http://dx.doi.org/10.1128/AEM.02440-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4725285PMC
February 2016

Clonal Clustering Using 10-Gene Multilocus Sequence Typing Reveals an Association Between Genotype and Listeria monocytogenes Maximum Growth Rate in Defined Medium.

Foodborne Pathog Dis 2015 Dec 23;12(12):972-82. Epub 2015 Oct 23.

2 Department of Veterinary and Microbiological Sciences, North Dakota State University , Fargo, North Dakota.

We used a 10-gene (10G) multilocus sequence typing scheme to investigate the diversity and phylogenetic distribution of 124 Listeria monocytogenes strains across major lineages, major serotypes, and seven epidemic clones that have been previously associated with outbreaks. The 124 isolates proved to be diverse, with a total of 81 sequence types (10G-STs) belonging to 13 clonal complexes (CCs), where all STs of the same CC differ from one another in up to 3 of the 10 alleles (named as 10G-triple-locus-variant-clonal-complexes [10G-TLV-CCs]). Phenotypic characterization for 105 of the 124 strains showed that L. monocytogenes had variable maximum growth rate (μ(max)) in a defined medium at 16°C, and classification by lineage or serotype was not able to reflect the genetic basis for the difference of this phenotype. Among the six major 10G-TLV-CCs, 10G-TLV-CC4 that included lineage I strains had significantly lower μ(max) (Tukey honestly significant difference adjusted [adj.] p < 0.05) compared to 10G-TLV-CC1 and 10G-TLV-CC3 that both comprised lineage II strains, indicating a distinct difference in growth of these L. monocytogenes isolates under nutrient-limited conditions among some of the CCs. However, the other three (10G-TLV-CC2, 6, and 10) of the six major 10G-TLV-CCs containing either lineage I or lineage II strains did not show significantly different μ(max) compared to the others (adj. p < 0.05). Our findings highlighted the importance of using molecular typing methods that can be used in evolutionary analyses as a framework for further understanding the phenotypic characteristics of subgroups of L. monocytogenes.
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http://dx.doi.org/10.1089/fpd.2015.2019DOI Listing
December 2015

Transcriptomic Analysis of the Adaptation of Listeria monocytogenes to Growth on Vacuum-Packed Cold Smoked Salmon.

Appl Environ Microbiol 2015 Oct 24;81(19):6812-24. Epub 2015 Jul 24.

Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, North Dakota, USA

The foodborne pathogen Listeria monocytogenes is able to survive and grow in ready-to-eat foods, in which it is likely to experience a number of environmental stresses due to refrigerated storage and the physicochemical properties of the food. Little is known about the specific molecular mechanisms underlying survival and growth of L. monocytogenes under different complex conditions on/in specific food matrices. Transcriptome sequencing (RNA-seq) was used to understand the transcriptional landscape of L. monocytogenes strain H7858 grown on cold smoked salmon (CSS; water phase salt, 4.65%; pH 6.1) relative to that in modified brain heart infusion broth (MBHIB; water phase salt, 4.65%; pH 6.1) at 7°C. Significant differential transcription of 149 genes was observed (false-discovery rate [FDR], <0.05; fold change, ≥2.5), and 88 and 61 genes were up- and downregulated, respectively, in H7858 grown on CSS relative to the genes in H7858 grown in MBHIB. In spite of these differences in transcriptomes under these two conditions, growth parameters for L. monocytogenes were not significantly different between CSS and MBHIB, indicating that the transcriptomic differences reflect how L. monocytogenes is able to facilitate growth under these different conditions. Differential expression analysis and Gene Ontology enrichment analysis indicated that genes encoding proteins involved in cobalamin biosynthesis as well as ethanolamine and 1,2-propanediol utilization have significantly higher transcript levels in H7858 grown on CSS than in that grown in MBHIB. Our data identify specific transcriptional profiles of L. monocytogenes growing on vacuum-packaged CSS, which may provide targets for the development of novel and improved strategies to control L. monocytogenes growth on this ready-to-eat food.
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http://dx.doi.org/10.1128/AEM.01752-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561693PMC
October 2015

VirR-Mediated Resistance of Listeria monocytogenes against Food Antimicrobials and Cross-Protection Induced by Exposure to Organic Acid Salts.

Appl Environ Microbiol 2015 Jul 24;81(13):4553-62. Epub 2015 Apr 24.

Department of Food Science, Cornell University, Ithaca, New York, USA Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, North Dakota, USA

Formulations of ready-to-eat (RTE) foods with antimicrobial compounds constitute an important safety measure against foodborne pathogens such as Listeria monocytogenes. While the efficacy of many commercially available antimicrobial compounds has been demonstrated in a variety of foods, the current understanding of the resistance mechanisms employed by L. monocytogenes to counteract these stresses is limited. In this study, we screened in-frame deletion mutants of two-component system response regulators associated with the cell envelope stress response for increased sensitivity to commercially available antimicrobial compounds (nisin, lauric arginate, ε-polylysine, and chitosan). A virR deletion mutant showed increased sensitivity to all antimicrobials and significantly greater loss of membrane integrity when exposed to nisin, lauric arginate, or ε-polylysine (P < 0.05). The VirR-regulated operon, dltABCD, was shown to be the key contributor to resistance against these antimicrobial compounds, whereas another VirR-regulated gene, mprF, displayed an antimicrobial-specific contribution to resistance. An experiment with a β-glucuronidase (GUS) reporter fusion with the dlt promoter indicated that nisin does not specifically induce VirR-dependent upregulation of dltABCD. Lastly, prior exposure of L. monocytogenes parent strain H7858 and the ΔvirR mutant to 2% potassium lactate enhanced subsequent resistance against nisin and ε-polylysine (P < 0.05). These data demonstrate that VirRS-mediated regulation of dltABCD is the major resistance mechanism used by L. monocytogenes against cell envelope-damaging food antimicrobials. Further, the potential for cross-protection induced by other food-related stresses (e.g., organic acids) needs to be considered when applying these novel food antimicrobials as a hurdle strategy for RTE foods.
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http://dx.doi.org/10.1128/AEM.00648-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4475887PMC
July 2015

The Listeria monocytogenes strain 10403S BioCyc database.

Database (Oxford) 2015 28;2015. Epub 2015 Mar 28.

Department of Food Science, Cornell University, Ithaca, NY 14853, USA.

Listeria monocytogenes is a food-borne pathogen of humans and other animals. The striking ability to survive several stresses usually used for food preservation makes L. monocytogenes one of the biggest concerns to the food industry, while the high mortality of listeriosis in specific groups of humans makes it a great concern for public health. Previous studies have shown that a regulatory network involving alternative sigma (σ) factors and transcription factors is pivotal to stress survival. However, few studies have evaluated at the metabolic networks controlled by these regulatory mechanisms. The L. monocytogenes BioCyc database uses the strain 10403S as a model. Computer-generated initial annotation for all genes also allowed for identification, annotation and display of predicted reactions and pathways carried out by a single cell. Further ongoing manual curation based on published data as well as database mining for selected genes allowed the more refined annotation of functions, which, in turn, allowed for annotation of new pathways and fine-tuning of previously defined pathways to more L. monocytogenes-specific pathways. Using RNA-Seq data, several transcription start sites and promoter regions were mapped to the 10403S genome and annotated within the database. Additionally, the identification of promoter regions and a comprehensive review of available literature allowed the annotation of several regulatory interactions involving σ factors and transcription factors. The L. monocytogenes 10403S BioCyc database is a new resource for researchers studying Listeria and related organisms. It allows users to (i) have a comprehensive view of all reactions and pathways predicted to take place within the cell in the cellular overview, as well as to (ii) upload their own data, such as differential expression data, to visualize the data in the scope of predicted pathways and regulatory networks and to carry on enrichment analyses using several different annotations available within the database.
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http://dx.doi.org/10.1093/database/bav027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4377088PMC
September 2015

Optimization of combinations of bactericidal and bacteriostatic treatments to control Listeria monocytogenes on cold-smoked salmon.

Int J Food Microbiol 2014 Jun 20;179:1-9. Epub 2014 Mar 20.

Department of Food Science, Cornell University, Ithaca, NY 14853, United States; Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108, United States. Electronic address:

Contamination of cold-smoked salmon by Listeria monocytogenes is a major concern for the seafood industry. The objectives of this study were to (i) determine the most effective bactericidal treatment for L. monocytogenes on salmon and (ii) optimize bactericidal and bacteriostatic treatment combinations to identify cost-effective treatments against L. monocytogenes on salmon. L. monocytogenes challenge trials were conducted in brain heart infusion (BHI) and on salmon disks that were supplemented with bactericidal compounds nisin (NIS), lauric arginate (LAE), ε-polylysine (EPL), and chitosan (CHIT). Subsequently, the most effective bactericidal compound was further tested by concurrent application of a blend of organic acid salts containing potassium lactate and sodium diacetate (PLSDA). L. monocytogenes populations were measured at 7 °C over 60 days, and initial cell density (N₀), maximum initial log reduction (Nr), lag phase (λ), maximum growth rate (μmax), and maximum cell density (Nmax) over 60 days storage were estimated. Time to recover to initial cell density (Tinitial) was also compared for combinations of bactericidal and bacteriostatic treatments. Varying degrees of antimicrobial effects were observed with bactericidal compounds in BHI. However, when tested on salmon, only NIS significantly decreased initial L. monocytogenes populations by approximately 2 log CFU/g, and reduced Nmax by approximately 1.5 logCFU/g compared to untreated control (CTRL). Nr achieved by the combined treatment of NIS and PLSDA was approximately 2 log CFU/g regardless of the presence of PLSDA, and a dose-dependent increase in Nr was observed with increasing NIS concentrations. PLSDA alone or in combination with 20 ppm NIS was most effective at delaying growth of L. monocytogenes. The greatest reduction in Nmax was observed with the combination of 20 ppm NIS and PLSDA; Nmax was 3.1 log CFU/g lower compared to CTRL. Comparison of Tinitial indicated that PLSDA with NIS can effectively retard growth of L. monocytogenes to its initial level (following initial reduction) and offers a cost benefit over using high concentrations of NIS alone. In summary, the combined application of NIS (for a bactericidal effect) and PLSDA (for a bacteriostatic effect) proved to be an effective treatment option to reduce initial levels as well as minimize subsequent growth of L. monocytogenes throughout the expected shelf-life of cold-smoked salmon.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2014.03.017DOI Listing
June 2014

Omics approaches in food safety: fulfilling the promise?

Trends Microbiol 2014 May 23;22(5):275-81. Epub 2014 Feb 23.

Department of Food Science, Cornell University, Ithaca, New York, USA. Electronic address:

Genomics, transcriptomics, and proteomics are rapidly transforming our approaches to the detection, prevention, and treatment of foodborne pathogens. Microbial genome sequencing in particular has evolved from a research tool into an approach that can be used to characterize foodborne pathogen isolates as part of routine surveillance systems. Genome sequencing efforts will not only improve outbreak detection and source tracking, but will also create large amounts of foodborne pathogen genome sequence data, which will be available for data-mining efforts that could facilitate better source attribution and provide new insights into foodborne pathogen biology and transmission. Although practical uses and application of metagenomics, transcriptomics, and proteomics data and associated tools are less prominent, these tools are also starting to yield practical food safety solutions.
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http://dx.doi.org/10.1016/j.tim.2014.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016976PMC
May 2014

Nisin resistance of Listeria monocytogenes is increased by exposure to salt stress and is mediated via LiaR.

Appl Environ Microbiol 2013 Sep 12;79(18):5682-8. Epub 2013 Jul 12.

Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, North Dakota, USA.

Growth of Listeria monocytogenes on refrigerated, ready-to-eat food is a significant food safety concern. Natural antimicrobials, such as nisin, can be used to control this pathogen on food, but little is known about how other food-related stresses may impact how the pathogen responds to these compounds. Prior work demonstrated that exposure of L. monocytogenes to salt stress at 7°C led to increased expression of genes involved in nisin resistance, including the response regulator liaR. We hypothesized that exposure to salt stress would increase subsequent resistance to nisin and that LiaR would contribute to increased nisin resistance. Isogenic deletion mutations in liaR were constructed in 7 strains of L. monocytogenes, and strains were exposed to 6% NaCl in brain heart infusion broth and then tested for resistance to nisin (2 mg/ml Nisaplin) at 7°C. For the wild-type strains, exposure to salt significantly increased subsequent nisin resistance (P < 0.0001) over innate levels of resistance. Compared to the salt-induced nisin resistance of wild-type strains, ΔliaR strains were significantly more sensitive to nisin (P < 0.001), indicating that induction of LiaFSR led to cross-protection of L. monocytogenes against subsequent inactivation by nisin. Transcript levels of LiaR-regulated genes were induced by salt stress, and lmo1746 and telA were found to contribute to LiaR-mediated salt-induced nisin resistance. These data suggest that environmental stresses similar to those on foods can influence the resistance of L. monocytogenes to antimicrobials such as nisin, and potential cross-protective effects should be considered when selecting and applying control measures for this pathogen on ready-to-eat foods.
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http://dx.doi.org/10.1128/AEM.01797-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754191PMC
September 2013

Efficacy of different antimicrobials on inhibition of Listeria monocytogenes growth in laboratory medium and on cold-smoked salmon.

Int J Food Microbiol 2013 Aug 29;165(3):265-75. Epub 2013 May 29.

Department of Food Science, Cornell University, Ithaca, NY 14853, USA.

Listeria monocytogenes is of particular concern in cold-smoked fish products as it can survive curing and cold-smoking, and can subsequently grow from low numbers to potentially hazardous levels during refrigerated storage. The purpose of this study was to (i) quantify the effects of organic acids, nisin, and their combinations on controlling L. monocytogenes growth on cold-smoked salmon at refrigeration temperatures, (ii) identify synergistic interactions of binary combinations of these antimicrobials, and (iii) determine if results from laboratory growth media can predict antimicrobial efficacy on cold-smoked salmon. Strains representing the genetic diversity of L. monocytogenes lineages I and II were grown in brain heart infusion (BHI) broth as well as on the surface of commercially produced wet-cured, cold-smoked salmon slices at 7°C. BHI broth and cold-smoked salmon were supplemented with sodium diacetate (SDA, 0.14% water phase (w.p.)), potassium lactate (PL, 2% w.p.), nisin (NI, 50ppm), and binary combinations of inhibitors at the same levels. Cell densities of L. monocytogenes were measured over time and used to calculate growth parameters, including initial cell density (N0), lag phase (λ), maximum growth rate (μmax), and maximum cell density (Nmax) for each antimicrobial treatment. N0 was significantly lowered by addition of NI with a similar average reduction on salmon (2.02±0.99 log(CFU/g)) and in BHI (1.51±0.83 log(CFU/ml)). Among all antimicrobial treatments, the combination of PL and SDA led to the greatest increase in λ both on salmon (7.1±3.6days) and in BHI (9.7±3.8days) when compared to the controls. The combination of PL and SDA had synergistic effects on increasing λ and lowering Nmax both in BHI and on salmon. Among all the treatments tested, the combination of NI and PL led to the greatest reductions in Nmax on salmon. We observed positive correlations between the growth parameters obtained from BHI broth and cold-smoked salmon, indicating that growth of L. monocytogenes in broth, to some extent, qualitatively reflected characteristics of growth on cold-smoked salmon under antimicrobial stresses. Results from BHI could quantitatively predict the variability of growth parameters obtained from salmon for lineage II strains, but not for lineage I strains. Although results from laboratory growth medium may not provide exact predictions of antimicrobial efficacy on cold-smoked salmon, they could be used to rapidly identify effective combinations for further examination on cold-smoked salmon.
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http://dx.doi.org/10.1016/j.ijfoodmicro.2013.05.018DOI Listing
August 2013

Correction: Wiedmann, M., et al. Exploration of the Role of the Non-Coding RNA SbrE in L. monocytogenes Stress Response. Int. J. Mol. Sci. 2013, 14, 378-393.

Int J Mol Sci 2013 May 3;14(5):9685. Epub 2013 May 3.

Department of Food Science, Cornell University, Ithaca, NY 14853, USA.

The original version of the paper in Section 3.8 reports that "The peptide mass tolerance and fragment mass tolerance values were 10 ppm and 30 mDa, respectively" [1] (p. 387). To help others who may want to use the same methods in the future, the authors would like to correct the wording to: "The peptide mass tolerance and fragment mass tolerance values were 30 ppm and 0.15 Da, respectively. In order to decrease the probability of false peptide identification, only peptides with significance scores above the identity threshold (at the 95% confidence interval), defined by Mascot probability analysis (www.matrixscience.com/help/scoring_help.html#PBM), were considered to be confidently identified and used for protein identification.  Furthermore, only proteins identified in all four iTRAQ samples through at least two peptides with a p-value of <0.05 (expectation value) were further analyzed". The authors would like to apologize for any inconvenience this may have caused to the readers of this journal.
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http://dx.doi.org/10.3390/ijms14059685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3676805PMC
May 2013

Exploration of the role of the non-coding RNA SbrE in L. monocytogenes stress response.

Int J Mol Sci 2012 12 24;14(1):378-93. Epub 2012 Dec 24.

Department of Food Science, Cornell University, Ithaca, NY 14853, USA.

SbrE is a ncRNA in Listeria monocytogenes, reported to be up-regulated by the alternative sigma factor σB. Initial quantitative RT-PCR (qRT-PCR) experiments on parent strains and isogenic ΔsigB strains demonstrated σB-dependent expression of SbrE across the four L. monocytogenes lineages and in L. innocua. Microarray and proteomics (MDLC/MS/MS with iTRAQ labeling) experiments with the L. monocytogenes parent strain and an isogenic ΔsbrE strain identified a single gene (lmo0636) and two proteins (Lmo0637 and Lmo2094) that showed lower expression levels in the ΔsbrE strain. qRT-PCR demonstrated an increase in SbrE transcript levels in stationary phase L. monocytogenes and in bacteria exposed to oxidative stress (mean log2 transcript levels 7.68 ± 0.57 and 1.70 ± 0.71 greater than in mid-log phase cells, respectively). However, no significant differences in growth or survival between the parent strain and ΔsbrE strain were confirmed under a variety of environmental stress conditions tested. Our data suggest that σB-dependent transcription of SbrE represents a conserved mechanism that contributes, across Listeria species, to fine-tuning of gene expression under specific environmental conditions that remain to be defined.
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http://dx.doi.org/10.3390/ijms14010378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3565269PMC
December 2012

Effect of curing method and freeze-thawing on subsequent growth of Listeria monocytogenes on cold-smoked salmon.

J Food Prot 2012 Sep;75(9):1619-26

Department of Food Science, Cornell University, Ithaca, New York 14853, USA.

The presence of the foodborne pathogen Listeria monocytogenes on cold-smoked salmon is a major concern for the seafood industry. Understanding processing and postprocessing handling factors that affect the ability of this pathogen to grow on cold-smoked salmon is critical for developing effective control strategies. In this study, we investigated the effect of curing method and freeze-thawing of cold-smoked salmon on (i) physicochemical properties and (ii) subsequent growth of genetically diverse strains of L. monocytogenes (inoculated after freeze-thawing) and endogenous lactic acid bacteria. The majority of the measured physicochemical properties were unaffected by freezing and thawing. Overall, wet-cured cold-smoked salmon had higher pH, water activity, and moisture, as well as lower fat, water-phase salt, and phenolic content compared with dry-cured cold-smoked salmon. The curing method and freeze-thawing did not affect growth of endogenous lactic acid bacteria. Freeze-thawing cold-smoked salmon prior to inoculation led to pronounced growth of L. monocytogenes at 7°C. The increase in cell density between days 0 and 30 was significantly (P = 0.0078) greater for cold-smoked salmon that was frozen and thawed prior to inoculation compared with nonfrozen cold-smoked salmon. On dry-cured, freeze-thawed cold-smoked salmon, L. monocytogenes had a lag phase ranging from 3.7 ± 0.1 to 11.2 ± 1.4 days compared with salmon that was wet cured and freeze-thawed, on which L. monocytogenes began to grow within 24 h. Variation in growth among L. monocytogenes strains was also observed, indicating the significance of assessing multiple strains. Further efforts to understand the impact of processing and postprocessing handling steps of cold-smoked salmon on the growth of genetically diverse L. monocytogenes will contribute to improved challenge study designs and data. This, in turn, will likely lead to more reliable and unbiased risk assessments and control measures.
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http://dx.doi.org/10.4315/0362-028X.JFP-11-561DOI Listing
September 2012

Listeria monocytogenes shows temperature-dependent and -independent responses to salt stress, including responses that induce cross-protection against other stresses.

Appl Environ Microbiol 2012 Apr 3;78(8):2602-12. Epub 2012 Feb 3.

Department of Food Science, Cornell University, Ithaca, New York, USA.

The food-borne pathogen Listeria monocytogenes experiences osmotic stress in many habitats, including foods and the gastrointestinal tract of the host. During transmission, L. monocytogenes is likely to experience osmotic stress at different temperatures and may adapt to osmotic stress in a temperature-dependent manner. To understand the impact of temperature on the responses this pathogen uses to adapt to osmotic stress, we assessed genome-wide changes in the L. monocytogenes H7858 transcriptome during short-term and long-term adaptation to salt stress at 7°C and 37°C. At both temperatures, the short-term response to salt stress included increased transcript levels of sigB and SigB-regulated genes, as well as mrpABCDEFG, encoding a sodium/proton antiporter. This antiporter was found to play a role in adaptation to salt stress at both temperatures; ΔmrpABCDEFG had a significantly longer lag phase than the parent strain in BHI plus 6% NaCl at 7°C and 37°C. The short-term adaptation to salt stress at 7°C included increased transcript levels of two genes encoding carboxypeptidases that modify peptidoglycan. These carboxypeptidases play a role in the short-term adaptation to salt stress only at 7°C, where the deletion mutants had significantly different lag phases than the parent strain. Changes in the transcriptome at both temperatures suggested that exposure to salt stress could provide cross-protection to other stresses, including peroxide stress. Short-term exposure to salt stress significantly increased H(2)O(2) resistance at both temperatures. These results provide information for the development of knowledge-based intervention methods against this pathogen, as well as provide insight into potential mechanisms of cross-protection.
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http://dx.doi.org/10.1128/AEM.07658-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318819PMC
April 2012

The transcriptional response of Listeria monocytogenes during adaptation to growth on lactate and diacetate includes synergistic changes that increase fermentative acetoin production.

Appl Environ Microbiol 2011 Aug 10;77(15):5294-306. Epub 2011 Jun 10.

405 Stocking Hall, Department of Food Science, Cornell University, Ithaca, NY 14853, USA.

The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of Listeria monocytogenes. Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two L. monocytogenes strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of L. monocytogenes.
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http://dx.doi.org/10.1128/AEM.02976-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147427PMC
August 2011

A small RNA controls expression of the chitinase ChiA in Listeria monocytogenes.

PLoS One 2011 Apr 18;6(4):e19019. Epub 2011 Apr 18.

Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019019PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078929PMC
April 2011

Transcriptomic and phenotypic analyses identify coregulated, overlapping regulons among PrfA, CtsR, HrcA, and the alternative sigma factors sigmaB, sigmaC, sigmaH, and sigmaL in Listeria monocytogenes.

Appl Environ Microbiol 2011 Jan 29;77(1):187-200. Epub 2010 Oct 29.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.

A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutation in a gene encoding a key regulatory protein, was used to characterize transcriptional networks in L. monocytogenes; the seven regulatory proteins addressed include all four L. monocytogenes alternative sigma factors (σ(B), σ(C), σ(H), and σ(L)), the virulence gene regulator PrfA, and the heat shock-related negative regulators CtsR and HrcA. Whole-genome microarray analyses, used to identify regulons for each of these 7 transcriptional regulators, showed considerable overlap among regulons. Among 188 genes controlled by more than one regulator, 176 were coregulated by σ(B), including 92 genes regulated by both σ(B) and σ(H) (with 18 of these genes coregulated by σ(B), σ(H), and at least one additional regulator) and 31 genes regulated by both σ(B) and σ(L) (with 10 of these genes coregulated by σ(B), σ(L), and at least one additional regulator). Comparative phenotypic characterization measuring acid resistance, heat resistance, intracellular growth in J774 cells, invasion into Caco-2 epithelial cells, and virulence in the guinea pig model indicated contributions of (i) σ(B) to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, σ(B), and CtsR to virulence-associated characteristics. Loss of the remaining transcriptional regulators (i.e., sigH, sigL, or sigC) resulted in limited phenotypic consequences associated with stress survival and virulence. Identification of overlaps among the regulons provides strong evidence supporting the existence of complex regulatory networks that appear to provide the cell with regulatory redundancies, along with the ability to fine-tune gene expression in response to rapidly changing environmental conditions.
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http://dx.doi.org/10.1128/AEM.00952-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3019704PMC
January 2011
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