The conjugative transfer of plasmid-mediated mobile colistin resistance gene, mcr-1, to Escherichia coli O157:H7 and Escherichia coli O104:H4 in nutrient broth and in mung bean sprouts.

The emergence of mobile colistin resistant gene (mcr-1) in Enterobacteriaceae has become a global public health concern. Dissemination of the mcr-1 gene through conjugation of bacteria associated with food may occur. This research investigated the transfer frequency of the mcr-1 gene among Escherichia coli in liquid media and during growth of mung bean sprouts. The donor strain E. coli NCTC 13846 (mcr-1 positive) and recipient strains of E. coli O157:H7 and E. coli O104:H4 were used. Mating experiments in vitro were conducted at 4, 25, and 37 °C for up to 36 h. The in vivo mating experiments (growing sprouts) were conducted in a sprout growth chamber with irrigation of 1 min/h over 6 days following inoculation of mung bean seeds with the donor and a recipient. The highest transfer frequencies in TSB media, 2.86E-07 and 3.24E-07, occurred at 37 °C after mating for 24 h for E. coli O104:H4 and E. coli O157:H7, respectively. Transconjugants were not detected in liquid media at 4 °C. Moreover, transfer frequency (5.68E-05 per recipient) of mcr-1 was greater during mung bean sprout growth for E. coli O104:H4 compared to E. coli O157:H7 (1.02E-05 per recipient) Day 3 to Day 6. This study indicates that the transfer of antibiotic resistant gene(s) among bacteria during mung bean sprout production may facilitate the spread of antibiotic resistant bacteria in the environment and to humans.

Influence of surface polysaccharides of Escherichia coli O157:H7 on plant defense response and survival of the human enteric pathogen on Arabidopsis thaliana and lettuce (Lactuca sativa).

This study aimed to determine the influence of bacterial surface polysaccharides (cellulose, colanic acid, and lipopolysaccharide; LPS) on the colonization or survival of Escherichia coli O157:H7 on plants and the plant defense response. Survival of E. coli O157:H7 were evaluated on Arabidopsis thaliana and romaine lettuce as a model plant and an edible crop (leafy vegetable), respectively. The population of the wild-type strain of E. coli O157:H7 on Arabidopsis plants and lettuce was significantly (P < 0.05) greater compared with the colanic acid-deficient and LPS-truncated mutants on day 1 and day 5 post-inoculation. This result indicates that colanic acid and LPS structures may contribute to the ability of bacterial survival or persistence on plants. The wild-type strain of E. coli O157:H7 produced approximately twice the amount (P < 0.05) of capsular polysaccharide (CPS) than the colanic acid and LPS-truncated mutants. The significantly lower production of CPS was associated with significantly greater (2-fold) expression of pathogenesis-related gene (PR1) compared with the wild-type and cellulose-deficient mutant (P < 0.05). Collectively, the results of this study may suggest that specific surface polysaccharides of E. coli O157:H7 differentially induce the plant defense response, consequently affecting the survival of the human pathogen on plants. The survival and persistence of E. coli O157:H7 was similar on Arabidopsis and lettuce regardless of day post-inoculation.

Ethnobotany of the genus Taraxacum-Phytochemicals and antimicrobial activity.

Plants belonging to the genus Taraxacum have been used in traditional healthcare to treat infectious diseases including food-borne infections. This review aims to summarize the available information on Taraxacum spp., focusing on plant cultivation, ethnomedicinal uses, bioactive phytochemicals, and antimicrobial properties. Phytochemicals present in Taraxacum spp. include sesquiterpene lactones, such as taraxacin, mongolicumin B, and taraxinic acid derivatives; triterpenoids, such as taraxasterol, taraxerol, and officinatrione; and phenolic derivatives, such as hydroxycinnamic acids (chlorogenic, chicoric, and caffeoyltartaric acids), coumarins (aesculin and cichoriin), lignans (mongolicumin A), and taraxacosides. Aqueous and organic extracts of different plant parts exhibit promising in vitro antimicrobial activity relevant for controlling fungi and Gram-positive and Gram-negative bacteria. Therefore, this genus represents a potential source of bioactive phytochemicals with broad-spectrum antimicrobial activity. However, so far, preclinical evidence for these activities has not been fully substantiated by clinical studies. Indeed, clinical evidence for the activity of Taraxacum bioactive compounds is still scant, at least for infectious diseases, and there is limited information on oral bioavailability, pharmacological activities, and safety of Taraxacum products in humans, though their traditional uses would suggest that these plants are safe.

Sanitizer efficacy in preventing cross-contamination of heads of lettuce during retail crisping.

This study was conducted to provide information regarding mitigation of cross-contamination through the use of sanitizer during crisping at retail outlets. Seven non-inoculated heads and one inoculated head (≈5 log CFU/g) of lettuce were placed into commercial sink filled with 76 L of tap water (TW), electrolyzed water (EW, free chlorine: 43 ± 6 ppm), lactic acid and phosphoric acid-based sanitizer (LPA, pH 2.89), or citric acid-based sanitizer (CA, pH 2.78) and soaked for 5 min. Two subsequent batches (eight non-inoculated heads per batch) were soaked in the same solution. Soaking with EW significantly reduced the population of S. enterica (2.8 ± 1.5 log CFU/g), E. coli O157:H7 (3.4 ± 1.1 log CFU/g), and L. monocytogenes (2.6 ± 0.7 log CFU/g) inoculated on Romaine lettuce compared to TW, LPA, and CA (p < 0.05). On Red leaf lettuce, EW significantly reduced populations of S. enterica and E. coli O157:H7, but not L. monocytogenes compared to other treatments. No significant difference was noted between TW, LPA, and CA in reducing foodborne pathogens (p > 0.05) or preventing cross-contamination. Soaking with EW prevented cross-contamination among lettuce heads and controlled bacterial populations in crisping water for three consecutive batches. EW may be an effective option as a sanitizer to minimizing the cross-contamination of leafy greens during the retail crisping.

Plants of the Genus Zingiber as a Source of Bioactive Phytochemicals: From Tradition to Pharmacy.

Plants of the genus Zingiber (Family Zingiberaceae) are widely used throughout the world as food and medicinal plants. They represent very popular herbal remedies in various traditional healing systems; in particular, rhizome of Zingiber spp. plants has a long history of ethnobotanical uses because of a plethora of curative properties. Antimicrobial activity of rhizome essential oil has been extensively confirmed in vitro and attributed to its chemical components, mainly consisting of monoterpene and sesquiterpene hydrocarbons such as α-zingiberene, ar-curcumene, ß-bisabolene and ß-sesquiphellandrene. In addition, gingerols have been identified as the major active components in the fresh rhizome, whereas shogaols, dehydrated gingerol derivatives, are the predominant pungent constituents in dried rhizome. Zingiber spp. may thus represent a promising and innovative source of natural alternatives to chemical food preservatives. This approach would meet the increasing concern of consumers aware of the potential health risks associated with the conventional antimicrobial agents in food. This narrative review aims at providing a literature overview on Zingiber spp. plants, their cultivation, traditional uses, phytochemical constituents and biological activities.

Potential transfer of extended spectrum ß-lactamase encoding gene, blashv18 gene, between Klebsiella pneumoniae in raw foods.

This study investigated the transfer frequency of the extended-spectrum ß-lactamase-encoding gene (blaSHV18) among Klebsiella pneumoniae in tryptic soy broth (TSB), pasteurized milk, unpasteurized milk, alfalfa sprouts and chopped lettuce at defined temperatures. All transconjugants were characterized phenotypically and genotypically. KP04(ΔKM) and KP08(ΔKM) isolated from seed sprouts and KP342 were used as recipients in mating experiments with K. pneumoniae ATCC 700603 serving as the donor. In mating experiments, no transconjugants were detected at 4 °C in liquid media or chopped lettuce, but detected in all media tested at 15 °C, 24 °C, and 37 °C. At 24 °C, the transfer of blaSHV18 gene occurred more frequently in alfalfa sprouts (5.15E-04 transconjugants per recipient) and chopped lettuce (3.85E-05) than liquid media (1.08E-05). On chopped lettuce, transconjugants were not detected at day 1 post-mating at 15 °C, but observed on day 2 (1.43E-05). Transconjugants carried the blaSHV18 gene transferred from the donor and the virulence gene harbored by recipient. More importantly, a class 1 integrase gene and resistance to tetracycline, trimethoprim/sulfamethoxazole were co-transferred during mating. These quantitative results suggest that fresh produce exposed to temperature abuse may serve as a competent vehicle for the spread of gene encoding for antibiotic resistance, having a potential negative impact on human health.

Differences in inactivation of Escherichia coli O157:H7 strains in ground beef following repeated high pressure processing treatments and cold storage.

High pressure processing (HPP) is a safe non-thermal processing method to effectively improve food safety. In this study, HPP treatment followed by cold storage was investigated to reduce Escherichia coli O157:H7 in ground beef. Experiments were conducted using ground beef contaminated with six E. coli O157:H7 strains one at a time or as a cocktail. Control and inoculated ground beef samples were HPP at 25 °C, 35 °C, and 45 °C, at 400 MPa and pre-determined number of pressure cycles totaling a holding time of 15 min. Optimum HPP parameters were 25 °C, 400 MPa at five pressure cycles of 3 min each which achieved a 5-log reduction of E. coli O157:H7 in ground beef. Storing HPP processed ground beef at 4 °C or -20 °C further decreased (P < 0.05) the E. coli O157:H7 population. An effective HPP treatment (5-log reduction) was developed that could be used post-processing to reduce the risk associated with E. coli O157:H7 contamination in ground beef.

Exposure of Escherichia coli O157:H7 to soil, manure, or water influences its survival on plants and initiation of plant defense response.

This study evaluated whether growth medium or exposure conditions influence the production of capsular polysaccharides (CPS) by Escherichia coli O157:H7, and whether changes in CPS impact plant defense responses, consequently affecting survival on plants. E. coli O157:H7 grown in Luria-Bertani (LB) broth supplemented with manure extracts showed an approximately 58% increase in CPS production compared to cells grown in LB medium alone. Levels of CPS were significantly higher for E. coli O157:H7 cells exposed to soil or manure extracts as compared to the non-exposed LB cultured control. Arabidopsis thaliana plants expressing ß-glucuronidase (GUS) under the control of the ß-1,3-glucanase (BGL2) promoter were used to investigate whether E. coli O157:H7 induces defense-related gene expression. Plants inoculated with E. coli O157:H7 grown in LB containing manure extracts or cells exposed to manure extracts exhibited 3-fold and 2-fold lower GUS activity, respectively, suggesting a limited plant defense response compared to plants inoculated with cells grown in LB. On day 5 post inoculation the population of E. coli O157:H7 grown in LB supplemented with manure on plants was significantly greater than the population of E. coli O157:H7 grown in LB medium alone. E. coli O157:H7 cells exposed to soil or manure exhibited greater survival on plants compared to LB-grown E. coli O157:H7. The results of this study underscore the need to consider medium composition and cultural conditions when conducting crop challenge studies.

Susceptibility of aged guinea pigs to repeated daily challenge with Listeria monocytogenes.

Epidemiological data suggests that certain groups such as the elderly are more susceptible to listeriosis than the general population. Repeated exposure to low levels of Listeria monocytogenes may increase the probability of infection. Increased susceptibility to infection in the elderly has been attributed in part to reduced activity of T cells. We investigated the impact of consecutive daily intragastric challenge with L. monocytogenes on the development of listeriosis within an aged guinea pig population. Approximately 63% of animals became infected following oral L. monocytogenes challenge with 10(4) CFU daily for 3 days, based on recovery of the pathogen from the liver or spleen. At day 4 postchallenge, 100% of animals were infected based on recovery of the bacterium in spleen and liver, decreasing to 14% by day 6 and then steadily increasing to 83% by day 13. During the 13-day postchallenge period, in the blood, numbers of total CD3(+) T cells decreased significantly; CD8(+) T-cell population underwent two shifts; CD4(+) T-cell population decreased and then increased. The results suggest that listerial infection can occur following repeated daily exposure to low levels of L. monocytogenes and that during infection, CD3(+) T-cell immune response may be depressed, potentially increasing susceptibility to other diseases.

The effect of crisping, misting, and storage temperature on the survival or growth of Listeria monocytogenes and natural psychrotrophic bacteria on romaine lettuce.

L. monocytogenes has been linked to fresh produce and detected in the retail environment. This study simulated the retail practices (crisping, misting, and storage) of unbagged whole heads of romaine lettuce to determine the growth of L. monocytogenes and natural psychrotrophic microflora. Three nalidixic acid-resistant strains of L. monocytogenes strains were inoculated to each head of lettuce (≈5 log10 CFU/g). For crisping, 24 heads of romaine lettuce were immersed in tap water or electrolyzed water (EW; free chlorine: 55 ppm) for 5 min, followed by holding at 5 °C for 2 h. The water-crisped (WC), EW crisped (EWC), or non-crisped (NC) lettuces were placed in a commercial refrigerated cabinet for misting at 5 °C. After 24-h misting, heads of lettuce were placed in perforated drain boxes with cover at 5 °C or 15 °C. The tap water and EW crisping achieved 1.3 and 2.9 log10 CFU/g reduction of L. monocytogenes, respectively. Approximately 1 log additional reduction of L. monocytogenes in the non-crisped lettuce was shown after misting (p < 0.05), but no significant effect of misting on the population of L. monocytogenes was observed on WC or EWC lettuces (p > 0.05). Regardless of the storage temperature or misting, L. monocytogenes populations remained significantly (p < 0.05) lower on EWC lettuce than NC and WC lettuce. On days 4 and 7 of storage, the natural psychrotrophic bacteria on lettuce stored at 5 °C was significantly lower than stored at 15 °C, and its population was not affected by crisping and misting. These provide insight into the influence of retail lettuce handling practices on the risk of L. monocytogenes.

Influences of photosensitizer curcumin on microbial survival and physicochemical properties of chicken during storage.

Curcumin is a natural plant derived antimicrobial, which was shown to inactivate or inhibit the growth of a broad spectrum of microorganisms through photodynamic inactivation. The purpose of the present study is to evaluate the influence of curcumin against commensal spoilage bacteria on chicken, foodborne pathogens, and the chicken skin pH and color. Chicken skin samples were immersed into water, photosensitizer curcumin (PSC), or peracetic acid (PAA). PSC samples were subsequently subjected to illumination by LEDs (430 nm). The PSC treatments did not inhibit the outgrowth of the four groups of spoilage bacteria evaluated. PSC treatment resulted in 2.9 and 1.5 log CFU/cm2 reduction of L. monocytogenes and Salmonella, respectively. Over a 10-d period, population of Salmonella remained significantly lower on PSC treated samples compared to other treatments. PSC treatment resulted in no significant changes in pH or color as compared to water treated samples. This research suggests PSC effectively controlled pathogen outgrowth on chicken without negatively influencing quality; and may be suitable for use in commercial chicken processing.

Influence of the plant defense response to Escherichia coli O157:H7 cell surface structures on survival of that enteric pathogen on plant surfaces.

Consumption of fresh and fresh-cut fruits and vegetables contaminated with Escherichia coli O157:H7 has resulted in hundreds of cases of illness and, in some instances, death. In this study, the influence of cell surface structures of E. coli O157:H7, such as flagella, curli fimbriae, lipopolysaccharides, or exopolysaccharides, on plant defense responses and on survival or colonization on the plant was investigated. The population of the E. coli O157:H7 ATCC 43895 wild-type strain was significantly lower on wild-type Arabidopsis plants than that of the 43895 flagellum-deficient mutant. The population of the E. coli O157:H7 43895 flagellum mutant was greater on both wild-type and npr1-1 mutant (nonexpressor of pathogenesis-related [PR] genes) plants and resulted in less PR gene induction, estimated based on a weak ß-glucuronidase (GUS) signal, than did the 43895 wild-type strain. These results suggest that the flagella, among the other pathogen-associated molecular patterns (PAMPs), made a substantial contribution to the induction of plant defense response and contributed to the decreased numbers of the E. coli O157:H7 ATCC 43895 wild-type strain on the wild-type Arabidopsis plant. A curli-deficient E. coli O157:H7 86-24 strain survived better on wild-type Arabidopsis plants than the curli-producing wild-type 86-24 strain did. The curli-deficient E. coli O157:H7 86-24 strain exhibited a GUS signal at a level substantially lower than that of the curli-producing wild-type strain. Curli were recognized by plant defense systems, consequently affecting bacterial survival. The cell surface structures of E. coli O157:H7 have a significant impact on the induction of differential plant defense responses, thereby impacting persistence or survival of the pathogen on plants.

Immune status and the development of Listeria monocytogenes infection in aged and young guinea pigs.

PURPOSE: Consuming even low numbers of the foodborne pathogen, Listeria monocytogenes, places the elderly at risk for severe illness. The impact of immunomodulation on the development of listerial infection within a young and aged population after low dose challenge with L. monocytogenes was investigated. METHODS: Animals received daily supplementation of vitamin E for a period of 21 days to promote immunomodulation, and were then orally challenged with 100 CFU of L. monocytogenes. Levels of CD8+, CD4+ and CD3+ T cells were used as markers to determine the influence of daily supplementation with vitamin E on immune response; the spleen and liver were harvested for microbiological analysis. RESULTS: Higher numbers of animals became infected in control groups than in vitamin E-treated group. During the post-challenge period, vitamin E-treated aged animals showed faster CD8+ T cell proliferation than control aged animals. CONCLUSION: Daily supplementation with vitamin E was more beneficial in young compared with aged animals in mitigating listerial infection. Results suggest exposure to even low numbers of L. monocytogenes can result in infection in both healthy young and aged populations.

Antimicrobial Resistance in the Food Chain: Trends, Mechanisms, Pathways, and Possible Regulation Strategies.

Antimicrobial resistance (AMR) remains of major interest for different types of food stakeholders since it can negatively impact human health on a global scale. Antimicrobial-resistant bacteria and/or antimicrobial resistance genes (transfer in pathogenic bacteria) may contaminate food at any stage, from the field to retail. Research demonstrates that antimicrobial-resistant bacterial infection(s) occur more frequently in low- and middle-income countries (LMICs) than in developed countries. Worldwide, foodborne pathogens are a primary cause of morbidity and mortality. The spread of pathogenic bacteria from food to consumers may occur by direct or indirect routes. Therefore, an array of approaches both at the national and international level to control the spread of foodborne pathogens and promote food safety and security are essential. Zoonotic microbes can spread through the environment, animals, humans, and the food chain. Antimicrobial drugs are used globally to treat infections in humans and animals and prophylactically in production agriculture. Research highlights that foods may become contaminated with AMR bacteria (AMRB) during the continuum from the farm to processing to retail to the consumer. To mitigate the risk of AMRB in humans, it is crucial to control antibiotic use throughout food production, both for animal and crop agriculture. The main inferences of this review are (1) routes by which AMRB enters the food chain during crop and animal production and other modes, (2) prevention and control steps for AMRB, and (3) impact on human health if AMR is not addressed globally. A thorough perspective is presented on the gaps in current systems for surveillance of antimicrobial use in food production and/ or AMR in the food chain.

Occurrence of Antibiotic-Resistant Staphylococcus spp. in Orange Orchards in Thailand.

BACKGROUND: The widespread indiscriminate application of antibiotics to food crops to control plant disease represents a potential human health risk. In this study, the presence of antibiotic-resistant staphylococci associated with workers and orange orchard environments was determined. A total of 20 orchards (orange and other fruits) were enrolled in the study. Trees in the orange orchards were treated with ampicillin on a pre-determined schedule. Environmental samples (n = 60) included soil, water, and oranges; 152 hand and nasal samples were collected from 76 healthy workers. Antibiotic susceptibility profiles were determined for all staphylococcal isolates. RESULTS: This investigation revealed that of the total Staphylococcus spp. recovered from the orange orchard, 30% (3/10) were resistant to erythromycin, 20% (2/10) were resistant to ampicillin, and 20% (2/10) resistant to both erythromycin and ampicillin. CONCLUSION: The application of antibiotics to orange trees in open production environments to halt the spread of bacterial disease presents risks to the environment and creates health concerns for Thai farmers using those agents. ARB on crops such as oranges may enter the global food supply and adversely affect public health.

Occurrence of antibiotic-resistant bacteria on hydroponically grown butterhead lettuce (Lactuca sativa var. capitata).

Antibiotics used during production of food crops to control plant diseases may result in selection of antibiotic-resistant bacteria and occurrence of antibiotic residues. The aim of this research was to evaluate the effect of antibiotics used in butterhead lettuce production on persistence of commensal microbiota. Butterhead lettuce were treated with antibiotics (oxytetracycline, gentamicin, and streptomycin) at different concentrations (100, 200, 300, 400 and 500 ppm) starting at 5 weeks' growth by spraying once daily for 4 weeks and harvesting 7 days after the final spray application. The population of total aerobic bacteria and antibiotic-resistant bacteria were determined. The results showed antibiotic usage significantly decreased bacterial populations on lettuce. Moreover, increased concentration of antibiotics resulted in significantly greater decrease in bacterial populations. At a concentration of 500 ppm, all antibiotics achieved an approximate 2 log CFU/g decrease in bacterial populations. A stable population (4 log CFU/g) of potentially antibiotic-resistant commensal microbiota were maintained throughout production. Screening for level of susceptibility indicated that bacteria exhibited greater resistance to oxytetracycline than gentamicin. In conclusion, application of antibiotics failed to eliminate commensal microbiota, demonstrating large populations of antibiotic-resistant bacteria reside on lettuce grown under conditions used in the present study. This is the first study focused on antibiotic usage on hydroponic systems. Results of this study suggest regulations directed at antibiotic use on food crops must be developed and implemented to control the selection and spread of antibiotic-resistant bacteria that present a global health concern.

Influence of product volume on water antimicrobial efficacy and cross-contamination during retail batch washing of lettuce.

Fresh produce shall be thoroughly washed at the retail level prior to serving to the consumer with potable water. Foodborne pathogens if present on a product may transfer to the wash water and cross-contaminate other products immersed in the water. Typically, an entire carton of lettuce (24 heads) is washed together increasing the likelihood of cross-contamination due to the close contact between each head. This study aimed to compare the effects of two wash batch volumes - "low" (8 heads) and "high" (24 heads) on the efficacy of two commercial water antimicrobials and cross-contamination. Red leaf lettuce was spot-inoculated (~5.0 log CFU/g) with Shiga toxin-producing Escherichia coli (STEC) and Listeria monocytogenes. In the first batch of washing, inoculated lettuce was washed with non-inoculated lettuce, followed by reuse of the water/antimicrobials twice washing only non-inoculated lettuce. Samples of inoculated and non-inoculated lettuce were collected to determine aerobic plate count (APC) as well as the populations of STEC and L. monocytogenes. Microbiological analysis of the wash water was also conducted. Wash volume (8 versus 24 lettuce heads) had limited effects on the antimicrobial activities of the interventions evaluated. Instead, high-volume wash increased the rate of cross-contamination between non-inoculated and inoculated lettuce, and cross-contamination of non-inoculated lettuce through wash water reuse. Retailers should consider volume of product processed per batch, reuse of wash water, and use of an appropriate water antimicrobial in consideration of mitigating potential product cross-contamination.

Transfer of Escherichia coli O157:H7 from soil, water, and manure contaminated with low numbers of the pathogen to lettuce plants.

The sources of contamination of leafy greens remain unclear, but it is evident that contaminated water, soil amendments, and wildlife likely contribute. The objective of the present study was to determine transfer of low numbers of Escherichia coli O157:H7 from soil, manure-amended soil, and water to growing lettuce plants. Lettuce plants, young (12 days of age) or mature (30 days of age), were grown in soil, manure-amended soil, or irrigated with water containing 10(1), 10(2), 10(3), or 10(4) CFU E. coli O157:H7 per g or ml. Harvested plants were processed to determine whether E. coli O157:H7 was associated with the entire plant or within internal locations. Young plants (12 days) were harvested at 1, 10, 20, and 30 days postexposure. No samples were positive for E. coli O157:H7 after direct plating of serial dilutions. Enrichment of all samples from young plants exposed to contaminated soil, manure-amended soil, and irrigation water demonstrated that approximately 21% (113 of 552) of plants were positive for E. coli O157:H7. Approximately 30% (36 of 120) of the mature plants initially irrigated with or grown in contaminated soil (including manure-amended soil) for 15 days were positive for E. coli O157:H7. Based on sterilization of surface tissue, E. coli O157:H7 was in protected locations of lettuce tissue. The results suggest that lettuce exposed to, and grown in the presence of, low numbers of E. coli O157:H7 may become contaminated and thus present a human health risk.

Influence of water antimicrobials and storage conditions on inactivating MS2 bacteriophage on strawberries.

Foodborne illnesses caused by norovirus contaminated fresh produce remain a food safety concern worldwide. In the present study, the impacts of commercial and home processing conditions of strawberries were evaluated for inactivation of the MS2 bacteriophage. MS2 was used as a surrogate of norovirus and was spot inoculated onto strawberries to achieve 6.6 log PFU/g. The inoculated strawberries were washed with tap water, electrolyzed water, or 50 ppm chlorine for 90 s prior to and after storage. After initial washing, the strawberries were separately stored at -20 °C and -80 °C for 30 days. Change in MS2 populations on strawberries was evaluated by plaque assay method on day 1, 15, and 30 for -20 °C and -80 °C groups. The results showed that washing strawberries prior to storage resulted in a significant decrease (approximately 1 log PFU/g) of MS2 population regardless of the treatment (p < 0.05). Frozen storage had minor effects on inactivating MS2, which resulted in approximately a 0.5 log PFU/g reduction at the end of storage. Washing frozen berries in electrolyzed water or 50 ppm chlorine on day 30 resulted in an additional 1 log PFU/g decrease in MS2 compared to water alone. These results suggest that washing strawberries with a chemical antimicrobial prior to and post frozen storage may enhance microbial safety.

Prevalence and Genetic Diversity of Cronobacter Species Isolated From Four Infant Formula Production Factories in China.

The aim of this study was to investigate the prevalence and genotypic characteristics of Cronobacter isolated from powdered infant formula (PIF) manufacturing facilities and to identify a potential source of contamination. A total of 42 Cronobacter isolates (5%) were detected in 835 environmental samples collected during the surveillance study. These isolates included C. sakazakii (n = 37), C. malonaticus (n = 3), and C. turicensis (n = 2). The isolates were divided into 14 sequence types (STs) by multi-locus sequence typing (MLST) and 21 pulsotypes (PTs) using pulsed-field gel electrophoresis (PFGE). The dominant C. sakazakii sequence types were ST3 (n = 12) and ST21 (n = 10), followed by ST136 (n = 6). The major PTs were PT22 (n = 12) and PT17 (n = 4) based on 100% similarity. Strains isolated from samples collected at the same production facility showed closer phylogenetic relation than those collected from distinct facilities. The result of extensive traceback sampling showed that PIF residues (PIF dust in production areas), fluid beds, drying areas, floors, and soil samples collected adjacent to the production facilities were the primary positive areas for Cronobacter. The present study outlines an effective approach to determine prevalence and genetic diversity of Cronobacter isolates associated with contamination of PIF.