Interlaboratory Evaluation of Enterococcus faecium NRRL B-2354 as a Salmonella Surrogate for Validating Thermal Treatment of Multiple Low-Moisture Foods.

ABSTRACT: This multi-institutional study assessed the efficacy of Enterococcus faecium NRRL B-2354 as a nonpathogenic Salmonella surrogate for thermal processing of nonfat dry milk powder, peanut butter, almond meal, wheat flour, ground black pepper, and date paste. Each product was analyzed by two laboratories (five independent laboratories total), with the lead laboratory inoculating (E. faecium or a five-strain Salmonella enterica serovar cocktail of Agona, Reading, Tennessee, Mbandaka, and Montevideo) and equilibrating the product to the target water activity before shipping. Both laboratories subjected samples to three isothermal treatments (between 65 and 100°C). A log-linear and Bigelow model was fit to survivor data via one-step regression. On the basis of D80°C values estimated from the combined model, E. faecium was more thermally resistant (P < 0.05) than Salmonella in nonfat dry milk powder (DEf-80°C, 100.2 ± 5.8 min; DSal-80°C, 28.9 ± 1.0 min), peanut butter (DEf-80°C, 133.5 ± 3.1 min; DSal-80°C, 57.6 ± 1.5 min), almond meal (DEf-80°C, 34.2 ± 0.4 min; DSal-80°C, 26.1 ± 0.2 min), ground black pepper (DEf-80°C, 3.2 ± 0.8 min; DSal-80°C, 1.5 ± 0.1 min), and date paste (DEf-80°C, 1.5 ± 0.0 min; DSal-80°C, 0.5 ± 0.0 min). Although the combined laboratory D80°C for E. faecium was lower (P < 0.05) than for Salmonella in wheat flour (DEf-80°C, 9.4 ± 0.1 min; DSal-80°C, 10.1 ± 0.2 min), the difference was ∼7%. The zT values for Salmonella in all products and for E. faecium in milk powder, almond meal, and date paste were not different (P > 0.05) between laboratories. Therefore, this study demonstrated the impact of standardized methodologies on repeatability of microbial inactivation results. Overall, E. faecium NRRL B-2354 was more thermally resistant than Salmonella, which provides support for utilizing E. faecium as a surrogate for validating thermal processing of multiple low-moisture products. However, product composition should always be considered before making that decision.

Cloning and Organelle Expression of Bamboo Mitochondrial Complex I Subunits Nad1, Nad2, Nad4, and Nad5 in the Yeast Saccharomyces cerevisiae.

Mitochondrial respiratory complex I catalyzes electron transfer from NADH to ubiquinone and pumps protons from the matrix into the intermembrane space. In particular, the complex I subunits Nad1, Nad2, Nad4, and Nad5, which are encoded by the nad1, nad2, nad4, and nad5 genes, reside at the mitochondrial inner membrane and possibly function as proton (H+) and ion translocators. To understand the individual functional roles of the Nad1, Nad2, Nad4, and Nad5 subunits in bamboo, each cDNA of these four genes was cloned into the pYES2 vector and expressed in the mitochondria of the yeast Saccharomyces cerevisiae. The mitochondrial targeting peptide mt gene (encoding MT) and the egfp marker gene (encoding enhanced green fluorescent protein, EGFP) were fused at the 5'-terminal and 3'-terminal ends, respectively. The constructed plasmids were then transformed into yeast. RNA transcripts and fusion protein expression were observed in the yeast transformants. Mitochondrial localizations of the MT-Nad1-EGFP, MT-Nad2-EGFP, MT-Nad4-EGFP, and MT-Nad5-EGFP fusion proteins were confirmed by fluorescence microscopy. The ectopically expressed bamboo subunits Nad1, Nad2, Nad4, and Nad5 may function in ion translocation, which was confirmed by growth phenotype assays with the addition of different concentrations of K+, Na+, or H+.

Stability of Listeria monocytogenes in non-fat dry milk powder during isothermal treatment and storage.

Dry dairy powder is a commonly used ingredient for ready-to-eat foods. It has been implicated in multiple foodborne outbreaks. Listeria monocytogenes can survive in low-moisture conditions for a long duration. However, there is no information on Listeria survival in dry milk powder during storage and thermal treatments. The objectives of this study were to examine the stability of L. monocytogenes in non-fat dry milk (NFDM) during extended storage and further analyze thermal resistance of L. monocytogenes in NFDM under different water activities (aw) and its thermal stability after 1-year storage. We observed approximately 1.75 and 2.93 log CFU/g reduction of L. monocytogenes in aw 0.25 NFDM over 1-year storage at 4 and 22 °C, respectively. Thermal resistance of L. monocytogenes was inversely related to aw, and the inactivation kinetic curves of L. monocytogenes in NFDM at target aw showed a log-linear trend under all tested conditions. For aw 0.25, 0.30, and 0.45 NFDM, the ranges of D-values, were 66.2-21.3, 33.5-9.4, and 14.6-4.3 min at 70, 75 and 80 °C, respectively. The z-values for L. monocytogenes in NFDM at aw 0.25-0.45 were 14.6-16.0 C°. Furthermore, the thermal stability of L. monocytogenes in aw 0.25 NFDM post 6-month or 12-month storage under refrigerated or ambient temperature did not deviate much from that in NFDM prior to the storage. Data indicated that a 60-min heat treatment at 80 °C resulted in ~ 5-log reduction of L. monocytogenes in NFDM of aw 0.30. This provides a promising intervention strategy to enhance bactericidal efficacy of thermal treatment while maintaining the quality of milk powder.

Microbial Safety of Dairy Manure Fertilizer Application in Raspberry Production.

Dairy manure, a by-product in the dairy industry, is also a potential source of nutrients for crops. However, improper application of biological soil amendments of animal origin can be a source of contamination with enteric foodborne pathogens. A 2-year field study was conducted to evaluate impacts of dairy manure fertilizer application on the microbial safety of red raspberry (Rubus idaeus L) production. Fertilizers, including a standard synthetic fertilizer (CON), straight lagoon raw manure (SL), anaerobically digested liquid effluent (DLE), compost (COM) and dairy manure-derived refined fertilizers including ammonium sulfate (AS) and phosphorous solid (PS), were randomly applied in quadruplicate to raspberry plots. Soil, fertilizer, foliar, and raspberry fruit samples were collected during the cropping season for the quantification of indicator microorganisms (total coliform and generic Escherichia coli) and detection of important foodborne pathogens (Shiga toxin-producing E. coli (STEC), Salmonella, and Listeria monocytogenes). Counts of total coliforms in soil were stable over the 2017 cropping season and were not impacted by fertilizer application. In 2018, total coliforms increased with season and soils treated with COM had a significantly higher coliform number than those treated with CON. Both total coliform and generic E. coli in raspberry fruit samples were below the detectable level (3 most probable number/g) regardless of fertilizer types. In both years, no STEC or L. monocytogenes was detected from any of the collected samples regardless of fertilizer treatments. However, Salmonella were detected in some of the fertilizers, including PS (2017), DLE (2018), and SL (2018), which were transferred to soil samples taken directly after application of these fertilizers. Salmonella were not detected in soil samples 2 or 4 months post fertilizer application, foliar, or raspberry fruit samples regardless of fertilizer applications. In summary, one-time application of raw dairy manure or dairy manure-derived fertilizers more than 4 months prior to harvest has no major impact on food safety of red raspberry (6 ft. tall) production in Lynden sandy loam under good agricultural practices.

Stabilization of Sir3 interactions by an epigenetic metabolic small molecule, O-acetyl-ADP-ribose, on yeast SIR-nucleosome silent heterochromatin.

In Saccharomyces cerevisiae, Sir proteins mediate heterochromatin epigenetic gene silencing. The assembly of silent heterochromatin requires histone deacetylation by Sir2, conformational change of SIR complexes, and followed by spreading of SIR complexes along the chromatin fiber to form extended silent heterochromatin domains. Sir2 couples histone deacetylation and NAD hydrolysis to generate an epigenetic metabolic small molecule, O-acetyl-ADP-ribose (AAR). Here, we demonstrate that AAR physically associates with Sir3 and that polySir3-AAR formation has a specific and essential role in the assembly of silent SIR-nucleosome pre-heterochromatin filaments. Furthermore, we show that AAR is capable of stabilizing binding of the Sir3 BAH domain to the Sir3 carboxyl-terminal region. Our data suggests that for the assembly of SIR-nucleosome pre-heterochromatin filament, the structural rearrangement of SIR-nucleosome is important and result in creating more stable interactions of Sir3, such as the inter-molecule Sir3-Sir3 interaction, and the Sir3-nucleosome interaction within the filaments. In conclusion, our results reveal the importance of AAR, indicating that it not only affects the conformational rearrangement of SIR complexes but also might function as a critical fine-tuning modulatory component of yeast silent SIR-nucleosome pre-heterochromatin by stabilizing the intermolecular interaction between Sir3 N- and C-terminal regions.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing.

Salmonella is capable of surviving in a low moisture environment for long periods. Once adapted to the xeric conditions, the thermal resistance of Salmonella is enhanced. Cocoa powder is a low water activity (aw) food (LawF) that is an essential component in a wide variety of desserts and ready-to-eat (RTE) foods and drinks. The aim of this study was to evaluate the desiccation and thermal resistance of Salmonella in cocoa powder, as well as to examine the suitability of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing. Natural unsweetened cocoa powder was inoculated with a 3-strain Salmonella cocktail or E. faecium and was equilibrated to aw 0.30 and 0.45 at room temperature, then subjected to isothermal treatments at 70-80 °C or 12-month storage at RT (room temperature, 22.0 ±â€¯0.5 °C, aw 0.30). At 70 and 80 °C, D-values of both Salmonella and E. faecium increased with decreasing aw. D-values of Salmonella at aw 0.30 cocoa powder were 46.2 ±â€¯4.7, 20.5 ±â€¯1.7, and 11.5 ±â€¯0.9 min at 70, 75 and 80 °C, respectively. Higher heat resistance of E. faecium in aw 0.30 cocoa powder was observed with D-values of 59.9 ±â€¯5.0, 28.9 ±â€¯1.8, and 16.1 ±â€¯1.4 min at 70, 75, and 80 °C, respectively. However, E. faecium demonstrated less heat resistance than Salmonella when aw was increased to 0.45. D-values for Salmonella at aw 0.45 were 31.6-7.0 min at 70-80 °C compared to 25.8-4.7 min for E. faecium. During 12 months of storage at RT, surviving E. faecium population in aw 0.30 cocoa powder was higher than that of the Salmonella cocktail; the population decreased by 1.39 and 3.75 logs, respectively. These findings indicate that the suitability of E. faecium as a surrogate organism for Salmonella is influenced by aw of cocoa powder. The aw correlates with thermal inactivation rates in both Salmonella and E. faecium, and should be considered as a significant contributor to the thermal resistance of Salmonella in cocoa powder.

A Major Facilitator Superfamily Transporter Regulated by the Stress-Responsive Transcription Factor Yap1 Is Required for Resistance to Fungicides, Xenobiotics, and Oxidants and Full Virulence in Alternaria alternata.

Alternaria alternata relies on the ability to produce a host-selective toxin and to detoxify reactive oxygen species to successfully colonize the host. An A. alternata major facilitator superfamily transporter designated AaMFS54 was functionally characterized by analysis of loss- and gain-of-function mutations to better understand the factors required for fungal pathogenesis. AaMFS54 was originally identified from a wild-type expression library after being subtracted with that of a mutant impaired for the oxidative stress-responsive transcription regulator Yap1. AaMFS54 contains 14 transmembrane helixes. Fungal mutant lacking AaMFS54 produced fewer conidia and increased sensitivity to many potent oxidants (potassium superoxide and singlet-oxygen generating compounds), xenobiotics (2,3,5-triiodobenzoic acid and 2-chloro-5-hydroxypyridine), and fungicides (clotrimazole, fludioxonil, vinclozolin, and iprodione). AaMFS54 mutant induced necrotic lesion sizes similar to those induced by wild-type on leaves of susceptible citrus cultivars after point inoculation with spore suspensions. However, the mutant produced smaller colonies and less fluffy hyphae on the affected leaves. Virulence assays on citrus leaves inoculated by spraying with spores revealed that AaMFS54 mutant induced less severe lesions than wild-type, indicating the requirement of AaMFS54 in pathogenesis. All defective phenotypes were restored in a strain re-acquiring a functional copy of AaMFS54. Northern blotting analysis revealed that the expression of AaMFS54 was suppressed by xenobiotics. The current studies indicate that the Yap1-mediated transporter plays a role in resistance to toxic oxidants and fungicides in A. alternata. The relationships of MFS transporters with other regulatory components conferring stress resistance and A. alternata pathogenesis are also discussed.

Fate of Listeria monocytogenes on Fresh Apples under Different Storage Temperatures.

Fresh apples are typically stored for up to 1 year commercially; different apple varieties require different storage temperatures to maintain their quality characteristics. There is sparse information available about Listeria monocytogenes survival on fresh apples under various storage temperatures. The objective of this study was to comprehensively evaluate the effect of storage temperature on apple fruit decay and L. monocytogenes survival. Unwaxed apple fruits of selected varieties (Fuji and Granny Smith) were dip inoculated in a three-strain L. monocytogenes cocktail to establish ∼3.5 and 6.0 Log10 CFU/apple. Twenty-four hours post-inoculation, apples were subjected to 1, 4, 10, or 22°C storage for up to 3 months. Apples under the different storage treatments were sampled at 1-, 4-, 7- and 14-day for short-term storage under all four tested temperatures, and 2-, 4-, 8-, and 12-week for long-term storage at 1, 4, and 10°C. A set of uninoculated and unwaxed apples were simultaneously subjected to the previously mentioned storage temperatures and sampled biweekly for their total bacterial count (TPC) and yeasts/molds (Y/M) count. During the 2-week short-term storage, L. monocytogenes population on organic Granny Smith apples stored at 1, 4, or 10°C was reduced by 0.2-0.3 Log. When apples were stored at 22°C, there was a 0.5-1.2 Log10 CFU/apple reduction 14-day post storage dependent on the initial inoculation level. During the 12-week cold storage under 1, 4, and 10°C, L. monocytogenes count on organic Granny Smith apples decreased by 0.5-1.5 Log10 CFU/apple for both inoculation levels. L. monocytogenes had similar survival pattern on conventional Granny Smith and Fuji apples with 0.8-2.0 Log10 CFU/apple reduction over a 3-month cold storage period. Interestingly, both TPC and Y/M count were stable regardless of apple variety or cultivation practice during the 12-week storage at all tested temperatures. In summary, while L. monocytogenes did not proliferate on apple surfaces during 12 weeks of refrigerated storage, only a limited reduction of L. monocytogenes was observed in this study. Therefore, the apple industry cannot rely on cold storage alone to control this pathogen. Additional interventions are needed to eradicate Listeria on fresh apples during long-term cold storage.

A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata.

Major Facilitator Superfamily (MFS) transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin), and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA). AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP) kinases, the 'two component' histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.

Calcineurin phosphatase and phospholipase C are required for developmental and pathological functions in the citrus fungal pathogen Alternaria alternata.

Excessive Ca(2+) or compounds interfering with phosphoinositide cycling have been found to inhibit the growth of the tangerine pathotype of Alternaria alternata, suggesting a crucial role of Ca(2+) homeostasis in this pathotype. The roles of PLC1, a phospholipase C-coding gene and CAL1, a calcineurin phosphatase-coding gene were investigated. Targeted gene disruption showed that both PLC1 and CAL1 were required for vegetative growth, conidial formation and pathogenesis in citrus. Fungal strains lacking PLC1 or CAL1 exhibited extremely slow growth and induced small lesions on calamondin leaves. Δplc1 mutants produced fewer conidia, which germinated at slower rates than wild-type. Δcal1 mutants produced abnormal hyphae and failed to produce any mature conidia, but instead produced highly melanized bulbous hyphae with distinct septae. Fluorescence microscopy using Fluo-3 dye as a Ca(2+) indicator revealed that the Δplc1 mutant hyphae emitted stronger cytosolic fluorescence, and the Δcal1 mutant hyphae emitted less cytosolic fluorescence, than those of wild-type. Infection assessed on detached calamondin leaves revealed that application of CaCl2 or neomycin 24 h prior to inoculation provided protection against Alt. alternata. These data indicate that a dynamic equilibrium of cellular Ca(2+) is critical for developmental and pathological processes of Alt. alternata.

Knockdown of PsbO leads to induction of HydA and production of photobiological H2 in the green alga Chlorella sp. DT.

Green algae are able to convert solar energy to H2 via the photosynthetic electron transport pathway under certain conditions. Algal hydrogenase (HydA, encoded by HYDA) is in charge of catalyzing the reaction: 2H(+)+2e(-)↔H2 but usually inhibited by O2, a byproduct of photosynthesis. The aim of this study was to knockdown PsbO (encoded by psbO), a subunit concerned with O2 evolution, so that it would lead to HydA induction. The alga, Chlorella sp. DT, was then transformed with short interference RNA antisense-psbO (siRNA-psbO) fragments. The algal mutants were selected by checking for the existence of siRNA-psbO fragments in their genomes and the low amount of PsbO proteins. The HYDA transcription and the HydA expression were observed in the PsbO-knockdown mutants. Under semi-aerobic condition, PsbO-knockdown mutants could photobiologically produce H2 which increased by as much as 10-fold in comparison to the wild type.

Cyclic AMP-dependent protein kinase A negatively regulates conidia formation by the tangerine pathotype of Alternaria alternata.

The necrotrophic fungal pathogen Alternaria alternata causes brown spot diseases in many citrus cultivars. The FUS3 and SLT2 mitogen-activated protein kinases (MAPK)-mediated signaling pathways have been shown to be required for conidiation. Exogenous application of cAMP to this fungal pathogen decreased conidia formation considerably. This study determined whether a cAMP-activated protein kinase A (PKA) is required for conidiation. Using loss-of-function mutations in PKA catalytic and regulatory subunit-coding genes, we demonstrated that PKA negatively regulates conidiation. Fungal mutants lacking PKA catalytic subunit gene (PKA ( cat )) reduced growth, lacked detectable PKA activity, and produced higher amounts of conidia compared to wild-type. Introduction of a functional copy of PKA ( cat ) into a null mutant partially restored PKA activity and produced wild-type level of conidia. In contrast, fungi lacking PKA regulatory subunit gene (PKA ( reg )) produced detectable PKA activity, exhibited severe growth reduction, formed swelling hyphal segments, and produced no mature conidia. Introduction of the PKA ( reg ) gene to a regulatory subunit mutant restored all phenotypes to wild type. PKA ( reg )-null mutants induced fewer necrotic lesions on citrus compared to wild-type, whereas PKA ( cat ) mutant displayed wild-type virulence. Overall, our studies indicate that PKA and FUS3-mediated signaling pathways apparently have very different roles in the regulation of conidia production and A. alternata pathogenesis in citrus.