Role of curli and plant cultivation conditions on Escherichia coli O157:H7 internalization into spinach grown on hydroponics and in soil (original) (raw)
Related papers
Journal of food protection, 2009
The incidence of foodborne outbreaks linked to fresh produce has increased in the United States. Particularly noteworthy was the 2006 Escherichia coli O157:H7 outbreak associated with prepackaged baby spinach. This study aimed to determine whether E. coli O157:H7 would be present in the aerial leaf tissue of a growing spinach plant when introduced at various plant maturities and different inoculum levels in a greenhouse setting. Spinach seeds of a commercial variety were sown in 8-in. (20.32-cm) pots. After seed germination, two levels (10(3) and 10(7) CFU/ml) of an E. coli O157:H7 green fluorescent protein-expressing strain were introduced into the plant growth media weekly for a total of five times. Inoculated spinach plants were examined weekly for the presence of E. coli O157:H7 on leaves and in surrounding growth media. Among 120 spinach plant samples examined for internal leaf contamination, only one yielded a positive result. Surface leaf contamination occurred occasionally a...
The incidence of foodborne outbreaks linked to fresh produce has increased in the United States. Particularly noteworthy was the 2006 Escherichia coli O157:H7 outbreak associated with prepackaged baby spinach. This study aimed to determine whether E. coli O157:H7 would be present in the aerial leaf tissue of a growing spinach plant when introduced at various plant maturities and different inoculum levels in a greenhouse setting. Spinach seeds of a commercial variety were sown in 8-in. (20.32-cm) pots. After seed germination, two levels (103 and 107 CFU/ml) of an E. coli O157:H7 green fluorescent protein–expressing strain were introduced into the plant growth media weekly for a total of five times. Inoculated spinach plants were examined weekly for the presence of E. coli O157:H7 on leaves and in surrounding growth media. Among 120 spinach plant samples examined for internal leaf contamination, only one yielded a positive result. Surface leaf contamination occurred occasionally and clustered between 3 and 5 weeks of age, but not among leaves younger than 3 weeks of age. On the other hand, when inoculated at the 107 CFU/ml level, the E. coli O157:H7 green fluorescent protein strain survived the entire cultivation period, although with gradually reduced levels. The experiments demonstrated that internalization of E. coli O157:H7 of growing spinach plant leaves under greenhouse conditions was a rare event, but surface contamination did occur, primarily when the plants reached 3 weeks of age. The study provided important data to further assess the association between spinach age and potential contamination of E. coli O157:H7.
Journal of Applied Microbiology, 2010
Aims: Survival of Escherichia coli O157:H7 and nonpathogenic E. coli on spinach leaves and in organic soil while growing spinach in a growth chamber was investigated.Methods and Results: Spinach plants were maintained in the growth chamber at 20°C (14 h) and 18°C (10 h) settings at 60% relative humidity. Five separate inocula, each containing one strain of E. coli O157:H7 and one nonpathogenic E. coli isolate were applied to individual 4-week-old spinach plants (cultivar ‘Whale’) grown in sandy soil. Leaf and soil inocula consisted of 100 μl, in 5 μl droplets, on the upper side of leaves resulting in 6·5 log CFU plant−1 and 1 ml in soil, resulting in 6·5 log CFU 200 g−1 soil per plant. Four replicates of each plant shoot and soil sample per inoculum were analysed on day 1 and every 7 days for 28 days for E. coli O157:H7 and nonpathogenic E. coli (by MPN) and for heterotrophic plate counts (HPC). Escherichia coli O157:H7 was not detected on plant shoots after 7 days but did survive in soil for up to 28 days. Nonpathogenic E. coli survived up to 14 days on shoots and was detected at low concentrations for up to 28 days. In contrast, there were no significant differences in HPC from days 0 to 28 on plants, except one treatment on day 7.Conclusions: Escherichia coli O157:H7 persisted in soil for at least 28 days. Escherichia coli O157:H7 on spinach leaves survived for less than 14 days when co-inoculated with nonpathogenic E. coli. There was no correlation between HPC and E. coli O157:H7 or nonpathogenic E. coli.Significance and Impact of the Study: The persistence of nonpathogenic E. coli isolates makes them possible candidates as surrogates for E. coli O157:H7 on spinach leaves in field trials.
Both growth chamber and field studies were conducted to investigate the potential for Escherichia coli O157:H7 to be internalized into leafy green tissue when seeds were germinated in contaminated soil. Internalized E. coli O157:H7 was detected by enrichment in both spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.) seedlings when seeds were germinated within the growth chamber in autoclaved and nonautoclaved soil, respectively, contaminated with E. coli O157:H7 at 2.0 and 3.8 log CFU/g, respectively. Internalized E. coli O157:H7 populations could be detected by enumeration within leafy green tissues either by increasing the pathogen levels in the soil or by autoclaving the soil. Attempts to maximize the exposure of seed to E. coli O157:H7 by increasing the mobility of the microbe either through soil with a higher moisture content or through directly soaking the seeds in an E. coli O157:H7 inoculum did not increase the degree of internalization. Based on responses obtained in growth chamber studies, internalization of E. coli O157:H7 surrogates (natural isolates of Shiga toxin–negative E. coli O157:H7 or recombinant [stx- and eae-negative] outbreak strains of E. coli O157:H7) occurred to a slightly lesser degree than did internalization of the virulent outbreak strains of E. coli O157:H7. The apparent lack of internalized E. coli O157:H7 when spinach and lettuce were germinated from seed in contaminated soil (ca. 3 to 5 log CFU/g) in the field and the limited occurrence of surface contamination on the seedlings suggest that competition from indigenous soil bacteria and environmental stresses were greater in the field than in the growth chamber. On the rare occasion that soil contamination with E. coli O157:H7 exceeded 5 log CFU/g in a commercial field, this pathogen probably would not be internalized into germinating leafy greens and/or would not still be present at the time of harvest.
Journal of Food Protection, 2013
Similar to phytopathogens, human bacterial pathogens have been shown to colonize the plant phylloplane. In addition to environmental factors, such as temperature, UV, relative humidity, etc., the plant cultivar and, specifically, the leaf blade morphological characteristics may affect the persistence of enteropathogens on leafy greens. This study was conducted to evaluate the effect of cultivar-dependent leaf topography and the role of strain phenotypic characteristics on Escherichia coli O157:H7 persistence on organic spinach. Spinach cultivars Emilia, Lazio, Space, and Waitiki were experimentally inoculated with the foodborne E. coli O157:H7 isolate EDL933 and its isogenic mutants deficient in cellulose, curli, or both curli and cellulose production. Leaves of 6-week-old plants were inoculated with 6.5 log CFU per leaf in a biosafety level 2 growth chamber. At 0, 1, 7, and 14 days, E. coli O157:H7 populations were determined by plating on selective medium and verified by laser scanning confocal microscopy. Leaf morphology (blade roughness and stoma density) was evaluated by low-temperature and variable-pressure scanning electron microscopy. E. coli O157:H7 persistence on spinach was significantly affected by cultivar and strain phenotypic characteristics, specifically, the expression of curli. Leaf blade roughness and stoma density influenced the persistence of E. coli O157:H7 on spinach. Cultivar Waitiki, which had the greatest leaf roughness, supported significantly higher E. coli O157:H7 populations than the other cultivars. These two morphological characteristics of spinach cultivars should be taken into consideration in developing intervention strategies to enhance the microbial safety of leafy greens.
Scientia Horticulturae, 2014
Irrigation water is an important vehicle for dissemination of human pathogens to plants. As contamination in an early stage of the production chain cannot necessarily be counteracted later, cultural measures to reduce the contamination risk need to be adopted during primary production. In a two-factorial greenhouse experiment, we studied the impact of inoculum density and the interval between irrigation and harvest on the prevalence of an inoculated gfp-tagged non-pathogenic strain of Escherichia coli O157:H7. The strain was inoculated with the irrigation water at a density of log 5.6, log 6.6 and log 7.6 CFU ml −1 into the phyllosphere of fully grown crops of rocket and spinach (BBCH 49). The crops were then harvested after 3, 24, 48 and 72 h. The introduced strain decreased exponentially in numbers within 72 h, to 49.6%, 52.6% and 50.6%, respectively, in the spinach and to 58.5%, 67.4% and 73.4% in the rocket. No differences were found in the number of the total viable count of aerobic bacteria and of Enterobacteriaceae as assessed on tryptic soy agar (TSA) and violet red bile dextrose agar (VRBD), respectively. Sequencing of the 16S rRNA genes of randomly selected isolates from VRBD were identified as Enterobacter cloaceae, Enterobacter ludwigii, Pantoea sp. and Raoultella planticola as the dominant Enterobacteriaceae species in the rocket and spinach phyllosphere. We found that cessation of irrigation for three days seems not to be an adequate sanitisation treatment to exclude the possibility of viable E. coli O157:H7 cells on spinach or rocket.
Food Microbiology, 2019
This study determined the variability in population uniformity of an applied mixture of attenuated E. coli O157:H7 (attEcO157) on spinach leaves as impacted by sampling mass and detection technique over spatial and temporal conditions. Opportunistically, the survival and distribution of naturally contaminating pathogenic E. coli O157:H7 (EcO157), in a single packaged lot following commercial postharvest handling and washing, was also evaluated. From the main study outcomes, differences in the applied inoculum dose of 100-fold, resulted in indistinguishable population densities of approximately Log 1.1 CFU g −1 by 14 days post-inoculation (DPI). Composite leaf samples of 150 g and the inclusion of the spinach petiole resulted in the greatest numerical sensitivity of detection of attEcO157 when compared to 25 and 150 g samples without petioles (P < 0.05). Differences in population density and protected-site survival and potential leaf internalization were observed between growing seasons and locations in California (P < 0.05). A Double Weibull model best described and identified two distinct populations with different inactivation rates of the inoculated attEcO157. Linear die-off rates varied between 0.14 and 0.29 Log/Day irrespective of location. Detection of EcO157-stx1-negative and stx2-positive, resulting from a natural contamination event, was observed in 11 of 26 quarantined commercial units of washed spinach by applying the 150 g sample mass protocol. The capacity to detect EcO157 varied between commercial test kits and non-commercial qPCR. Our findings suggest the need for modifications to routine pathogen sampling protocols employed for lot acceptance of spinach and other leafy greens.
Both spinach and lettuce were grown to harvest, cut, and then regrown after spraying the cut shoots with irrigation water contaminated with Escherichia coli O157:H7. Plant tissue was collected on the day of spraying and again 2 and 14 days later for analysis of total and internalized E. coli O157:H7 populations. Internalization of E. coli O157:H7 occurred on the day of spraying, and larger populations were internalized as the level in the spray increased. Tissue repair was slow and insufficient to prevent infiltration of E. coli O157:H7; internalized E. coli O157:H7 in shoots cut 5 days prior to exposure to E. coli O157:H7contaminated water were not significantly different from levels in shoots cut on the same day of spraying with contaminated water (P . 0.05). Two days after spraying plants with a high level of E. coli O157:H7 (7.3 log CFU/ml), levels of internalized E. coli O157:H7 decreased by ca. 2.6 and 1.3 log CFU/g in Tyee and Bordeaux spinach, respectively, whereas populations of internalized E. coli O157:H7 decreased very little (ca. 0.4 log CFU/g) in lettuce plants that had been sprayed either on the same day as cutting or 1 day after cutting. When cut plants were sprayed with irrigation water at a lower contamination level (4.5 log CFU/ml), internalized E. coli O157:H7 was not detected in either spinach or lettuce plants 2 days later and therefore would not likely be of concern when the crop was harvested.
International Journal of Food Microbiology, 2010
Recent outbreaks of food-borne illnesses associated with the consumption of fresh produce have increased attention on irrigation water as a potential source of pathogen contamination. A better understanding of the behaviour of enteric pathogens introduced into agricultural systems during irrigation will aid in risk assessments and support the development of appropriate farm-level water management practices. For this reason, the survival dynamics of two nalidixic acid resistant strains of Escherichia coli after their spray inoculation into the phyllosphere and soil of field spinach were examined over two growing seasons. E. coli strains NAR, an environmental isolate, and DM3n, a non-pathogenic serotype O157:H7, were applied at rates of 10 4 to 10 7 cfu/100 ml to the fully developed spinach plants that arose subsequent to the harvesting of their upper leafy portions for commercial purposes (secondary-growth plants). After 72 h, E. coli on spinach were reduced by 3-5 logs. Culturable E. coli were recovered from plants up to 6 days post-inoculation. Survival in soil was greater than in the phyllosphere. Under ambient conditions, the mean 72 h first order decay constant computed by Chick's Law was 0.1 h −1. Although light reduction studies indicated UV irradiation negatively influenced the persistence of E. coli, a simple relationship between UV exposure and phyllosphere E. coli densities could not be established. E. coli introduced to the leafy portions of spinach via spray irrigation displayed rapid declines in their culturability under the open environmental conditions experienced during this study. A 6 day period between the last irrigation and harvest would minimize the risks of E. coli survival in the spinach phyllosphere. E. coli NAR was identified as a possible surrogate for the O157:H7 strain, DM3n.
2011
The California lettuce and leafy greens industry has adopted the Leafy Greens Marketing Agreement (LGMA), which allows for 126 most-probable-number (MPN) Escherichia coli per 100 ml in irrigation water. Repeat irrigation of baby spinach plants with water containing E. coli O157:H7 and different levels of total organic carbon (TOC) was used to determine the epiphytic survival of E. coli O157:H7. Three irrigation treatments (0 ppm of TOC, 12 or 15 ppm of TOC, and 120 or 150 ppm of TOC) were prepared with bovine manure containing E. coli O157:H7 at either low (0 to 1 log CFU/100 ml) or high (5 to 6 log CFU/ 100 ml) populations, and sprayed onto baby spinach plants in growth chambers by using a fine-mist airbrush. MPN and direct plating techniques were used to determine the E. coli O157:H7 populations on the aerial plant tissue. Plants irrigated with high E. coli O157:H7 populations, regardless of TOC levels, showed a 3-log reduction within the first 24 h. Low levels of E. coli O157:H7 were observed for up to 16 days on all TOC treatments, ranging from 76.4 MPN per plant (day 1) to 0.40 MPN per plant (day 16). No viable cells were detected on spinach tissue 24 h after irrigation with water containing fewer than 126 CFU/ 100 ml E. coli O157:H7. Under growth chamber conditions in this study, E. coli O157:H7 populations in irrigation water that complies with the LGMA standards will not persist for more than 24 h when applied onto foliar surfaces of spinach plants.