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Enterobacteriaceae species have caused foodborne illnesses through consumption of a variety of foods, including infant foods. A total of 915 samples representing 4 types of samples, were collected from 7 powdered infant formula factories in Heilongjiang Province, China. All samples were processed for enumeration of Enterobacteriaceae and isolation of Cronobacter sakazakii, which were detected using the ISO enrichment procedure and a chromogenic medium. Multi-locus sequence typing (MLST), and pulsed field gel electrophoresis (PFGE) were applied to characterize the C. sakazakiiisolates to trace back the original source of contamination. Samples were considered Enterobacteriaceae positive at 10 CFU/g in powdered samples, 1 CFU/mL in water samples, and 10 CFU/100 cm2or larger surface area. Enterobacteriaceae positive rate were 0/35 water samples associated with production, 11/56 raw materials, 147/761 environmental samples and 3/63 finished products respectively. C. sakazakiistrains were isolated from 7.0% of environmental samples followed by 1.6% of the finished products. A total of 20 C. sakazakiiisolates were recovered in Factory 4, 1 strain derived from a final product and 19 strains recovered from the environment representing 4 MLST sequence types and 10 PFGE pulse types. Most environmental sites positive for C. sakazakiihad higher counts of Enterobacteriaceae, except for 11 sites where Enterobacteriaceae did not reach minimal levels for reporting a positive sample. The present study, provides insight to the correlation between the prevalence of Enterobacteriaceae and contamination of C. sakazakiiin powdered infant formula factories; there is no one-to-one relation between number of Enterobacteriaceae and C. sakazakii. Identifying environmental sites positive for C. sakazakiiand/or Enterobacteriaceae will aid in development of appropriate corrective measures to improve environmental hygiene and enhance safety of powdered infant formula.

AbstractEpidemiological 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 monocytogenesmay 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. monocytogeneson the development of listeriosis within an aged guinea pig population. Approximately 63% of animals became infected following oral L. monocytogeneschallenge with 104CFU 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. monocytogenesand that during infection, CD3+T-cell immune response may be depressed, potentially increasing susceptibility to other diseases.

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 coliO157: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 101, 102, 103, or 104CFU E. coliO157:H7 per g or ml. Harvested plants were processed to determine whether E. coliO157: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. coliO157: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. coliO157: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. coliO157:H7. Based on sterilization of surface tissue, E. coliO157: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. coliO157:H7 may become contaminated and thus present a human health risk.

The efficacy of commercially available antimicrobials for fresh produce—electrolyzed water (EW; around 60 mg/L of free chlorine), a combination of lactic acid and phosphoric acid-based (LPA) and citric acid-based (CA)—was compared with tap water (TW) in preventing cross-contamination during the soaking step of crisping at a retail setting. A bunch of cilantro ((103.7±14.9) g/bunch) was inoculated with a three-strain cocktail of nalidixic acid-resistant Salmonella enterica(S. enterica) and Escherichia coli(E. coli) O157:H7, S. entericaand Listeria monocytogenes(L. monocytogenes), or E. coliO157:H7 and L. monocytogenes(around 5.0 log CFU/g). One inoculated and seven non-inoculated cilantro bunches were soaked in 76 L of TW, EW, LPA, and CA for 5 min. Two additional soakings, each with eight bunches of non-inoculated cilantro, were performed in the same soaking water. To determine the cross-contamination of inoculated foodborne pathogens via soaking water, the cilantro samples and soaking water following each soaking step were subjected to microbiological analyses using selective media supplemented with nalidixic acid (100 µg/mL). During the first soaking, significantly greater reductions in Salmonella((2.9±0.5) log CFU/g), E. coliO157:H7 ((3.0±0.1) log CFU/g), and L. monocytogenes((2.7±0.3) log CFU/g) on cilantro were achieved with EW compared to soaking with TW, LPA, and CA (P<0.05). Cross-contamination of foodborne pathogens from inoculated cilantro to non-inoculated cilantro was completely mitigated by EW during three subsequent soaking events. With the exception of TW soaking water, no inoculated foodborne pathogens were detected in the 100 mL soaking water of EW, CA, and LPA collected. Including an appropriate concentration of chemical antimicrobial in water during the soaking step of crisping aids in mitigating cross-contamination of foodborne pathogen(s) in cilantro bunches.

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/cm2reduction of L. monocytogenesand Salmonella, respectively. Over a 10-day period, population of Salmonellaremained 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.

Foodborne pathogens may exist as endophytes of growing plants. The internalization of Escherichia coliO157:H7 or other foodborne pathogens in growing lettuce plants may be independent of microbial factors. Mature lettuce plants were surface irrigated with E. coliO157:H7 or with FluoSpheres (fluorescent microspheres) and harvested 1, 3, and 5 days post-exposure. FluoSpheres were utilized as a bacterial surrogate. Microscopic examination of root, stem, and leaf tissue sections revealed that FluoSpheres were internalized into growing plants. Laser scanning confocal microscopy revealed that FluoSpheres were present within the root tissue and leaf stem tissue. The presence of FluoSpheres in internal portions of stem and leaf tissue suggests transport of the spheres from the root upward into the edible tissue. The level of uptake of FluoSpheres and E. coliO157:H7 was quantified using filtration. Numbers of FluoSpheres and E. coliO157:H7 cells in plant tissue were similar. The entry of E. coliO157:H7 into lettuce plants may be a passive event because the concentration of FluoSpheres was similar to that of the pathogen.

Chloramphenicol-resistant mutants ofEscherichia coliO157:H7 (n= 52) demonstrated multiple resistance to tetracycline, nalidixic acid, and ciprofloxacin. Approximately half of the mutants approached the clinical level of resistance to the antibiotic tetracycline. Mutants demonstrated a wide range of sensitivity to each antibiotic; for tetracycline, some isolates demonstrated a twofold increase, whereas others demonstrated a 15-fold increase in resistance. Continuous exposure to chloramphenicol did not affect the growth of mutants, suggesting that mutation does not have a negative effect on cell survival. Complementation experiments with a functional marRrestored antibiotic susceptibility of selected mutants to levels similar to wild-type strains, suggesting that marmutation was responsible for the decrease in sensitivity. The multiple antibiotic resistance (mar) operon is a global regulator controlling intrinsic resistance toward structurally and functionally unrelated antibiotics and other noxious agents. Antimicrobial use in both human and agricultural practice should be administered in a manner to prevent selection of resistant mutants.

Irrigation water collected at farms growing crops for human consumption was artificially contaminated with E. coliO157: H7 and used to irrigate lettuce plants. Plants in a growth chamber were spray irrigated either once or intermittently with water contaminated with 102or 104CFU of E. coliO157:H7 per ml and were then sampled over a 30-day period. Only plants exposed to 102CFU/ml on day 1 did not harbor the pathogen at the end of the sampling period. All other treatments resulted in contaminated plants at harvest. Plants irrigated with 104CFU/ml contained high levels (up to 5 log CFU/g) of the pathogen at harvest. The results obtained in this study underscore the assertion that spray irrigation (the application of water directly to plant leaves) is linked to the contamination of crops and suggest that repeated exposure increases the E. coliO157:H7 level on the plant.

In this study, the transmission of Escherichia coliO157:H7 to lettuce plants through spray and surface irrigation was demonstrated. For all treatments combined, the number of plants testing positive following a single exposure to E. coliO157: H7 through spray irrigation (29 of 32 plants) was larger than the number testing positive following surface irrigation (6 of 32 plants). E. coliO157:H7 persisted on 9 of 11 plants for 20 days following spray irrigation with contaminated water. Immersion of harvested lettuce heads for 1 min in a 200 ppm chlorine solution did not eliminate all E. coliO157:H7 cells. The results of this study suggest that regardless of the irrigation method used, crops can become contaminated; therefore, the irrigation of food crops with water of unknown microbial quality should be avoided.

Laser scanning confocal microscopy (LSCM) was used to observe the interaction of SalmonellaStanley with alfalfa sprouts. The green fluorescent protein (gfp) gene was integrated into the chromosome of SalmonellaStanley for constitutive expression, thereby eliminating problems of plasmid stability and loss of signal. Alfalfa seeds were inoculated by immersion in a suspension of SalmonellaStanley (ca. 107CFU/ml) for 5 min at 22°C. Epifluorescence microscopy demonstrated the presence of target bacteria on the surface of sprouts. LSCM demonstrated bacteria present at a depth of 12μm within intact sprout tissue. An initial population of ca. 104CFU/g seed increased to 7.0 log CFU/g during a 24-h germination period and then decreased to 4.9 log CFU/g during a 144-h sprouting period. Populations of SalmonellaStanley on alfalfa seeds decreased from 5.2 to 4.1 log CFU/g and from 5.2 to 2.8 log CFU/g for seeds stored 60 days at 5 and 22°C, respectively. The efficacy of 100, 200, 500, or 2,000 ppm chlorine in killing SalmonellaStanley associated with sprouts was determined. Treatment of sprouts in 2,000 ppm chlorine for 2 or 5 min caused a significant reduction in populations of SalmonellaStanley. Influence of storage on SalmonellaStanley populations was investigated by storing sprouts 4 days at 4°C. The initial population (7.76 log CFU/g) of SalmonellaStanley on mature sprouts decreased (7.67 log CFU/g) only slightly. Cross-contamination during harvest was investigated by harvesting contaminated sprouts, then directly harvesting noncontaminated sprouts. This process resulted in the transfer of ca. 105CFU/g SalmonellaStanley to the noncontaminated sprouts.

In farm and food processing environmentsEscherichia coli O157:H7 can survive for days to months. In this study, we investigated the influence of starvation, pH (pH 4 or 7), temperature (4, 10 and 22C), and exposure to chlorine (50, 100, 150, 200 ppm) on culturability ofE. coli O157:H7. The culturable cell population in PBS decreased to an undetectable level at 4C and 10C, but not at 22C during the experimental period. Cells suspended in PBS adjusted to pH 4 lost culturability more rapidly than at pH 7. Culturable cells were not detectable after exposure to chlorine, however, viable cell populations in the range of 1 – 2 log remained stable for 5 days. During loss of culturability two distinct morphological cell populations emerged; typical rod shaped and coccoid shaped cells. In this study, independent of exposure to chlorine, conditions of low temperature and low pH had the greatest influence on entry ofE. coli O157:H7 into a VNC state.

Staphylococcus aureusis a frequent cause of mastitis in dairy cows. However, pathogenesis of the infection has not been completely defined. We report the invasion of two strains of S. aureusinto a bovine mammary epithelial cell line and a bovine mammary epithelial cell primary culture. Invasion of S. aureusinto bovine mammary cells was time-dependent. Transmission electron microscopy of bovine mammary cells invaded by S. aureusshowed intracellular replication of the bacterium within membrane-bound vacuoles. Invasion was reduced significantly when bovine mammary epithelial cells were treated with inhibitors of F-actin microfilament polymerization but not when these cells were treated with inhibitors of microtubule formation. Results indicated that S. aureusis capable of invading and replicating inside bovine mammary epithelial cells. Data also suggested that S. aureusinvasion of bovine mammary epithelial cells requires active participation of specific components of the cytoskeleton of the epithelial cell.

Capsicumgenus (Solanaceae) is native to the Americas. Today, it is an important agricultural crop cultivated around the world, not only due to its economic importance, but also for the nutritional value of the fruits. Among their phytochemical constituents, capsaicinoids are characteristic and responsible of the pungency of sharp-tasting cultivars. Moreover, Capsicumand capsaicinoids (mainly, capsaicin) have been largely studied because of their health benefits. Thus, this study reviews the scientific knowledge about Capsicumspp. and their phytochemicals against cancer, diabetes, gastrointestinal diseases, pain, and metabolic syndrome, as well as their antioxidant and antimicrobial activity. These bioactivities can be the basis of the formulation of functional ingredients and natural preservatives containing Capsicumextracts or isolated compounds.

The Lavandulagenus, belonging to the Lamiaceae, includes 39 species, with nearly 400 registered cultivars. Lavandulaare worldwide plants that occur over the Mediterranean, Europe, North Africa, southwest Asia to southeast India. Lavandulaplants have been used since ancient time to flavor and preserved food, to treat diseases including wound healing, sedative, antispasmodic, microbial and viral infections. Numerous researches have described the chemical composition and the primary components of lavender oils are the monoterpenoids (linalool, linalyl acetate, 1,8-cineole, β-ocimene, terpinen-4-ol, and camphor), sesquiterpenoids (β-caryophyllene and nerolidol) and other terpenoid compounds (e.g., perillyl alcohol). The high concentrations of linalyl acetate make them attractive in perfumery, flavoring, cosmetics and soap industries. Currently, data on the antimicrobial activity of lavender plants have been scientifically confirmed. Indeed, lavender essential oils possess wide spectra of biological activities such as antispasmodic, carminative, analgesic, sedative, hypotensive, antiseptic, antimicrobial, antifungal, antidiuretic and general tonic action. In addition, clinical studies support their uses as treatment of health conditions. However, further clinical studies are necessary to define the magnitude of the efficacy, mechanisms of action, optimal doses, long-term safety, and, potential side effects of lavender plants.