Persistence of Listeria innocua on Fresh Apples during Long-Term Controlled Atmosphere Cold Storage with Postharvest Fungal Decay.

ABSTRACT: Recent apple-related recall and outbreak events have exposed a need for better food safety controls along the supply chain. Following harvest, apples can be stored under a controlled atmosphere for up to 1 year after harvest before packing and distribution, making the crop susceptible to many opportunities for contamination that increase the quantity of postharvest losses. Botrytis cinerea and Penicillium expansum cause significant rot-associated losses to the apple industry. These fungi can colonize and destroy apple tissue as storage duration increases, which may also impact the growth of saprophytic foodborne pathogens like Listeria monocytogenes. Thus, the objective of this study was to observe population changes of Listeria innocua as a surrogate for L. monocytogenes on apples inoculated with B. cinerea or P. expansum under long-term controlled atmosphere cold storage conditions to identify the effect of postharvest mold growth on growth patterns of a microorganism relevant to food safety. 'Gala' and 'WA 38' apples (n = 1,080) were harvested, treated with pyrimethanil, and inoculated with L. innocua only or with L. innocua and one of the mold species on wounded and unwounded portions of the apple equator. Apples were treated with 1-methylcyclopropene and stored at a controlled atmosphere (2 kPa O2, 1 kPa CO2, 1°C) for 1 week and 1, 3, 6, 9, and 11 months before enumeration. After 3 months, L. innocua consistently fell below the limit of detection (2.35 Log CFU/g), and samples were enriched following a modified Bacteriological Analytical Manual method with PCR confirmation. Listeria persistence was dependent on the storage duration and type of fungal contamination (P ≤ 0.05). Surface wounding may impact these trends, depending on the apple variety. Prevalence of L. innocua was greater in Gala apples. Future studies should more closely examine the interactions on the fruit surface that occur during the seemingly critical time frame of 3 to 6 months in storage.

Prevalence of Salmonella enterica in Flies on a Diversified Cattle and Fresh Produce Farm across Two Growing Seasons.

ABSTRACT: Flies are a vector for spreading foodborne pathogens pertinent to fresh produce, such as Shiga toxigenic Escherichia coli and Salmonella; however, most studies focus on concentrated animal feeding operations, which do not reflect low-density animal farming practices that often adjoin fruit and vegetable acreage. In this study, we determined the prevalence of Salmonella in flies collected biweekly on an integrated animal and produce operation over two growing seasons. Eleven of 889 pooled samples tested positive for Salmonella. Flies from the Calliphoridae, Muscidae, Sarcophagidae, and Tachinidae families were associated with Salmonella carriage, but fly family was not a significant factor for isolation of Salmonella (P = 0.303). Fly species were a significant factor (P = 0.026), with five Pentacricia aldrichii pools testing positive for Salmonella. With the exception of single specimen isolation, prevalence ranged from 2.2 to 15.2%. With the exception of the Tachinidae family, these results reflect a strong association of flies that are commonly associated with feces or are pests of animals. Trap location was not significantly associated with isolation of Salmonella-positive flies (P = 0.236). Overall, the population of flies was not as abundant as studies conducted with produce grown close to concentrated animal feeding operations, indicating a reduced risk of transmission; however, similar to these studies, fly families that are commonly isolated from fecal and decaying matter were most frequently associated with Salmonella isolation. Further work is warranted to elucidate the foodborne pathogen transmission rates to produce and subsequent survival over time.

Increased transcription of the phosphate-specific transport system of Escherichia coli O157:H7 after exposure to sodium benzoate.

Sodium benzoate is a widely used food antimicrobial in drinks and fruit juices. A microarray study was conducted to determine the transcriptional response of Escherichia coli O157:H7 to 0.5% (wt/vol) sodium benzoate. E. coli O157:H7 grown in 150 ml of Luria-Bertani broth was exposed to 0% (control) and 0.5% sodium benzoate. Each treatment was duplicated and sampled at 0 (immediately after exposure), 5, 15, 30, and 60 min. Total RNA was extracted and analyzed with E. coli 2.0 Gene Chips. Significant ontology categories affected by sodium benzoate exposure were determined with JProGO software. The phosphate-specific transport (Pst) system transports inorganic phosphate into bacterial cells, under phosphate-limited conditions. The Pst system was found to be highly upregulated. Increased expression of the Pst system was observed after the short 5 min of exposure to sodium benzoate; pstS, pstA, pstB, and pstC genes were upregulated more than twofold (linear scale) at 5, 15, 30, and 60 min. Increased expression of several other efflux systems, such as AcrAB-TolC, was also observed. The Pst system may act as an efflux pump under these stress-adapted conditions, as well as increase transport of phosphorus to aid in DNA, RNA, ATP, and phospholipid production. Understanding adaptations of Escherichia coli O157:H7 under antimicrobial exposure is essential to better understand and implement methods to inhibit or control its survival in foods.

Transcriptomic Behavior of Salmonella enterica Newport in Response to Oxidative Sanitizers.

ABSTRACT: Agricultural water is a known source of contamination to fresh produce and can contain foodborne pathogens including Salmonella enterica, pathogenic Escherichia coli, Listeria monocytogenes, and Campylobacter jejuni. To mitigate such risks, antimicrobial agents such as hypochlorites and peroxyacetic acid (PAA) can be applied to in-line irrigation systems as well as to water used in postharvest washing. Although these compounds are effective and widely used, some pathogenic bacteria adapt to survive exposure. RNA sequencing was used to analyze the Salmonella Newport transcriptome after exposure to sodium hypochlorite (NaOCl) and PAA in a simulated agricultural water system. Overall cellular adaptive response was determined quantitatively as a function of overall gene expression of the >4,000 genes in the Salmonella Newport genome. Differentially expressed genes ranged from 11 due to 10-ppm NaOCl treatment, 316 due to 20-ppm NaOCl treatment, 1,719 due to 10-ppm PAA treatment, and 2,010 due to 20-ppm PAA treatment compared with that of the controls (water only). Differentially expressed transcripts included cellular functions such as biosynthesis, degradation, energy generation, and nonmetabolically linked functions. Oxidative exposure upregulated genes associated with key virulence, attachment, and gene transfer. Amino acid biosynthesis was upregulated due to NaOCl exposure but primarily downregulated when Salmonella Newport was exposed to PAA. Slight upregulation occurred in nucleoside and nucleotide biosynthesis, a known DNA repair mechanism seen during exposure to sanitizers. Our results indicate that Salmonella Newport reacts differently when exposed to NaOCl versus PAA, despite oxidative activity being the primary modes of antimicrobial action of both compounds.

Prevalence, Persistence, and Diversity of Listeria monocytogenes and Listeria Species in Produce Packinghouses in Three U.S. States.

ABSTRACT: Listeria monocytogenes has emerged as a food safety concern for several produce commodities. Although L. monocytogenes contamination can occur throughout the supply chain, contamination from the packinghouse environment represents a particular challenge and has been linked to outbreaks and recalls. This study aimed to investigate the prevalence, persistence, and diversity of L. monocytogenes and other species of Listeria in produce packinghouses. A longitudinal study was performed in 11 packinghouses (whose commodities included microgreen, peach, apple, tomato, broccoli, cauliflower, and cucumber) in three U.S. states. In each packinghouse, 34 to 47 sites representing zones 2 to 4 were selected and swabbed. Packinghouses were visited four times over the packing season, and samples were tested for Listeria by following the U.S. Food and Drug Administration's Bacteriological Analytical Manual methods. Presumptive Listeria-positive isolates were confirmed by PCR. Species and allelic type (AT) were identified by sigB sequencing for up to eight isolates per sample. Among 1,588 samples tested, 50 (3.2%), 42 (2.7%), and 10 (0.6%) samples were positive for L. monocytogenes only, Listeria spp. (excluding L. monocytogenes) only, and both L. monocytogenes and Listeria spp., respectively. Five species of Listeria (L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, and L. marthii) were identified, and L. monocytogenes was the most prevalent species. The 102 Listeria-positive samples yielded 128 representative isolates (i.e., defined as isolates from a given sample with a different AT). Approximately 21% (21 of 102) of the Listeria-positive samples contained two or more ATs. A high AT diversity (0.95 Simpson's diversity index) was observed among Listeria isolates. There were three cases of L. monocytogenes or Listeria spp. repeated isolation (site testing positive at least twice) based on AT data. Data from this study also support the importance of drain and moisture management, because Listeria were most prevalent in samples collected from drain, cold storage, and wet nonfood contact surface sites.

Real-time reverse-transcriptase polymerase chain reaction for the rapid detection of Salmonella using invA primers.

Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to help control the spread of disease. Reverse-transcriptase polymerase chain reaction (RT-PCR) can detect the presence of mRNA (shorter half-life than DNA) with greater potential for detecting viable pathogens. The chromosomally located invA gene required for host invasion by Salmonella is widely used for detection of this pathogen by PCR. Detection of Salmonella was undertaken by real-time RT-PCR (rt-RT-PCR) using newly designed invA gene primers to develop a sensitive and specific assay. Salmonella serovars Typhimurium and Enteritidis were grown (7.68 log(10) CFU/mL) in Luria-Bertani broth overnight at 37 degrees C, and RNA was extracted, followed by rt-RT-PCR with and without SYBR green I and agarose gel electrophoresis. All experiments were replicated at least thrice. Detection for both serovars using traditional RT-PCR was lower ( approximately 10(5) CFU/mL) than rt-RT-PCR (10(3) CFU/mL) by gel electrophoresis. Melt curve analysis showed melt temperatures at 87.5 degrees C with Ct values from 12 to 15 for up to 10(3) CFU/mL and improved to 10(2) CFU/mL after further optimization. Further, addition of RNA internal amplification control constructed using in vitro transcription with a T7 RNA polymerase promoter, to the RT-PCR assay also gave detection limits of 10(2) CFU/mL. Cross-reactivity was not observed against a panel of 21 non-Salmonella bacteria. Heat-inactivated (autoclaved) Salmonella showed faint or no detection by rt-RT-PCR or gel electrophoresis. This method has potential to be applied for the detection of Salmonella serovars in fresh produce and the simultaneous detection of foodborne viral (RNA viruses) and bacterial pathogens in a multiplex format.

Transcription analysis of stx1, marA, and eaeA genes in Escherichia coli O157:H7 treated with sodium benzoate.

Expression of the multiple antibiotic resistance (mar) operon causes increased antimicrobial resistance in bacterial pathogens. The activator of this operon, MarA, can alter expression of >60 genes in Escherichia coli K-12. However, data on the expression of virulence and resistance genes when foodborne pathogens are exposed to antimicrobial agents are lacking. This study was conducted to determine transcription of marA (mar activator), stx1 (Shiga toxin 1), and eaeA (intimin) genes of E. coli O157:H7 EDL933 as affected by sodium benzoate. E. coli O157:H7 was grown in Luria-Bertani broth containing 0 (control) and 1% sodium benzoate at 37 degrees C for 24 h, and total RNA was extracted. Primers were designed for hemX (209 bp; housekeeping gene), marA (261 bp), and eaeA (223 bp) genes; previously reported primers were used for stx1. Tenfold dilutions of RNA were used in a real-time one-step reverse transcriptase PCR to determine transcription levels. All experiments were conducted in triplicate, and product detection was validated by gel electrophoresis. For marA and stx1, real-time one-step reverse transcriptase PCR products were detected at a 1-log-greater dilution in sodium benzoate-treated cells than in control cells, although cell numbers for each were similar (7.28 and 7.57 log CFU/ml, respectively). This indicates a greater (albeit slight) level of their transcription in treated cells than in control cells. No difference in expression of eaeA was observed. HemX is a putative uroporphyrinogen III methylase. The hemX gene was expressed at the same level in control and treated cells, validating hemX as an appropriate housekeeping marker. These data indicate that stx1 and marA genes could play a role in pathogen virulence and survival when treated with sodium benzoate, whereas eaeA expression is not altered. Understanding adaptations of E. coli O157:H7 during antimicrobial exposure is essential to better understand and implement methods to inhibit or control survival of this pathogen in foods.

Atmospheric plasma inactivation of foodborne pathogens on fresh produce surfaces.

A study was conducted to determine the effect of one atmosphere uniform glow discharge plasma (OAUGDP) on inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on apples, cantaloupe, and lettuce, respectively. A five-strain mixture of cultured test organisms was washed, suspended in phosphate buffer, and spot inoculated onto produce (7 log CFU per sample). Samples were exposed inside a chamber affixed to the OAUGDP blower unit operated at a power of 9 kV and frequency of 6 kHz. This configuration allows the sample to be placed outside of the plasma generation unit while allowing airflow to carry the antimicrobial active species, including ozone and nitric oxide, onto the food sample. Cantaloupe and lettuce samples were exposed for 1, 3, and 5 min, while apple samples were exposed for 30 s, 1 min, and 2 min. After exposure, samples were pummeled in 0.1% peptone water-2% Tween 80, diluted, and plated in duplicate onto selective media and tryptic soy agar and incubated as follows: E. coli O157:H7 (modified eosin methylene blue) and Salmonella (xylose lysine tergitol-4) for 48 h at 37 degrees C, and L. monocytogenes (modified Oxford medium) at 48 h for 32 degrees C. E. coli O157:H7 populations were reduced by >1 log after 30-s and 1-min exposures and >2 log after a 2-min exposure. Salmonella populations were reduced by >2 log after 1 min. Three- and 5-min exposure times resulted in >3-log reduction. L. monocytogenes populations were reduced by 1 log after 1 min of exposure. Three- and 5-min exposure times resulted in >3- and >5-log reductions, respectively. This process has the capability of serving as a novel, nonthermal processing technology to be used for reducing microbial populations on produce surfaces.

Antimicrobial Efficacy of an Array of Essential Oils Against Lactic Acid Bacteria.

The essential oils of clove bud, cinnamon bark and thyme, and their individual compounds including allyl isothiocyanate (AIT), carvacrol, cinnamaldehyde, cinnamic acid, eugenol, and thymol were initially assessed for antimicrobial activity against 9 lactic acid bacteria (LAB) species. Carvacrol and thymol were the most inhibitory with MICs of 0.1% (v/v and w/v, respectively). Cinnamaldehyde, cinnamon bark oil, clove bud oil, eugenol, and thyme oil were moderately inhibitive (MICs = 0.2% v/v), while cinnamic acid required a concentration of 0.5% (w/v). AIT was not effective with MICs in excess of concentrations tested (0.75% v/v). The bactericidal capability of the oil components carvacrol, cinnamaldehyde, eugenol, and thymol were further examined against Pediococcus acidilactici, Lactobacillus buchneri, and Leuconostoc citrovorum. Thymol at 0.1% (w/v) was bactericidal against L. citrovorum (>4-log reduction), but resulted in a 2-log CFU/mL reduction against L. buchneri and P. acidilactici. Cinnamaldehyde at 0.2% to 0.25% (v/v) was effective against L. citrovorum, L. buchneri, and P. acidilactici, resulting in a >2-log reduction. All 3 organisms were susceptible to 0.2% carvacrol with >3-log reduction observed after exposure for 6 h. Eugenol was the least effective. Concentrations of 0.2% and 0.25% (v/v) were needed to achieve an initial reduction in population, >3-log CFU/mL after 6 h exposure. However, at 0.2%, P. acidilactici and L. buchneri recovered to initial populations in 48 to 72 h. Results indicate essential oils have the capacity to inactivate LAB that are commonly associated with spoilage of shelf stable low-acid foods.

Antimicrobial Activity of Cinnamaldehyde, Carvacrol, and Lauric Arginate against Salmonella Tennessee in a Glycerol-Sucrose Model and Peanut Paste at Different Fat Concentrations.

The objective of this study was to investigate the antimicrobial activities of carvacrol, cinnamaldehyde, and lauric arginate (LAE) against Salmonella in a low water activity (aw ) glycerol-sucrose model and in peanut paste with different fat concentrations. Salmonella Tennessee was inoculated into the model and the low fat (<5%) and high fat (50%) peanut paste adjusted to aw 1.0, 0.7, 0.5, and 0.3 and with or without cinnamaldehyde, carvacrol, or LAE. The survival of the bacterium over 3 or 5 days at 25°C was evaluated. Reduced aw alone decreased the viable population over time, with the highest reduction at the lowest aw. In the glycerol-sucrose model, all antimicrobial agents significantly reduced the population over time (P < 0.05) compared with the controls. LAE was more lethal than the essential oil components, reducing the population to undetectable levels by day 2 for all aw. Cinnamaldehyde was more effective than carvacrol at aw 0.5 and 0.3 (2.7- to 2.9-log versus 0.39- to 1.97-log reductions on day 3). In low-fat peanut paste, none of the antimicrobial agents inhibited growth of the pathogen at aw 1.0. However, inactivation was enhanced at reduced aw. Cinnamaldehyde and LAE both reduced the pathogen population to undetectable levels on day 5 at the highest concentration tested (ca. 10 times higher than that in the glycerol-sucrose model). Inactivation efficacy of all antimicrobial agents was greatly decreased but not eliminated in 50% fat peanut paste. Results suggest that the test antimicrobial agents were effective under low aw conditions, but significantly higher concentrations are needed for potential food applications, and fat concentration can negatively impact the efficacy of these antimicrobial agents.

Naturally occurring antimicrobials for minimally processed foods.

Natural antimicrobials are gaining increased interest from researchers and food manufacturers alike seeking to discover label-friendly alternatives to the widely implemented synthetic compounds. Naturally occurring antimicrobials can be applied directly to food to protect food quality, extend food shelf life by inhibiting or inactivating spoilage microorganisms, and improve food safety by inhibiting or inactivating food-borne pathogens. There are a great number of natural antimicrobials derived from animal, plant, and microbial sources. This manuscript reviews their efficacy against spoilage and pathogenic organisms, their methods of evaluation, and their application in various foods as well as the development of novel delivery systems and incorporation with other hurdles.

Microbial ecology of foodborne pathogens associated with produce.

The recent recognition of fresh fruits and vegetables as major vehicles of foodborne illness has led to increased research on mechanisms by which enteric pathogens contaminate and persist on and in this non-host environment. Interactions between foodborne pathogens and plants as well among the naturally occurring microbial communities contribute to endophytic and epiphytic colonization. Scientific findings are just beginning to elucidate the mechanisms that contribute to colonization of produce. This review addresses current knowledge as well as future research needed to increase our understanding of the microbial ecology of enteric pathogens on fruits and vegetables.

Inactivation of foodborne pathogens using a one atmosphere uniform glow discharge plasma.

This study was conducted to determine the efficacy of a one atmosphere uniform glow discharge plasma (OAUGDP) for inactivation of foodborne pathogens and to evaluate the influence of growth temperature, pH, and culture age on their inactivation. Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Salmonella Enteritidis, Vibrio parahaemolyticus, Yersinia enterocolitica, and Shigella flexneri were evaluated. Three-strain mixtures of each bacterium were inoculated (6-7 log CFU/cm(2)) onto microscope slides containing nonselective agar media adjusted to pH 5 or 7. Samples were exposed to plasma for 0-240 sec immediately, or after incubation for 24 h at 10 degrees C or 35 degrees C. After exposure, the agar was removed from the slides and pummeled in 0.1% peptone water with a stomacher, serially diluted, surface plated onto nonselective media, and incubated at 35 degrees C. Exposure time, pH, incubation temperature, and culture age affected survival of all pathogens exposed to plasma (P < 0.05). The greatest reduction of pathogens generally occurred during the initial exposure time of 30 or 90 sec. Pathogens incubated for 24 h before exposure were more resistant than those exposed immediately after inoculation. Incubation at 35 degrees C before exposure resulted in greater resistance to plasma inactivation than incubation at 10 degrees C. No appreciable differences between gram-positive and gram-negative pathogens were observed, although the spore-forming B. cereus was more resistant to plasma than non-spore-formers. These findings support the potential for plasma treatment of foods or surfaces for pathogen reduction. Increased sensitivity of pathogens to plasma at reduced pH and temperature is encouraging, since these conditions are applicable to many foods during processing, handling, and storage.