The endophytic lifestyle of Escherichia coli O157:H7: quantification and internal localization in roots.

The foodborne pathogen Escherichia coli O157:H7 is increasingly associated with fresh produce (fruit and vegetables). Bacterial colonization of fresh produce plants can occur to high levels on the external tissue but bacteria have also been detected within plant tissue. However, questions remain about the extent of internalization, its molecular basis, and internal location of the bacteria. We have determined the extent of internalization of E. coli O157:H7 in live spinach and lettuce plants and used high-resolution microscopy to examine colony formation in roots and pathways to internalization. E. coli O157:H7 was found within internal tissue of both produce species. Colonization occurred within the apoplast between plant cells. Furthermore, colonies were detected inside the cell wall of epidermal and cortical cells of spinach and Nicotiana benthamiana roots. Internal colonization of epidermal cells resembled that of the phytopathogen Pectobacterium atrosepticum on potato. In contrast, only sporadic cells of the laboratory strain of E. coli K-12 were found on spinach, with no internal bacteria evident. The data extend previous findings that internal colonization of plants appears to be limited to a specific group of plant-interacting bacteria, including E. coli O157:H7, and demonstrates its ability to invade the cells of living plants.

AFM study of the differential inhibitory effects of the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) against Gram-positive and Gram-negative bacteria.

(-)-Epigallocatechin-3-gallate (EGCG), a main constituent of tea catechins, affects Gram-positive and Gram-negative bacteria differently; however, the underlying mechanisms are not clearly understood. Atomic force microscopy (AFM) was used to compare morphological alterations in Gram-positive and Gram-negative bacteria induced by EGCG and by H(2)O(2) at sub-minimum inhibitory concentrations (MICs). EGCG initially induced aggregates in the cell envelopes of Staphylococcus aureus and eventually caused cell lysis, which was not observed in cells treated with H(2)O(2). It initially induced nanoscale perforations or microscale grooves in the cell envelopes of Escherichia coli O157:H7 which eventually disappeared, similar to E. coli cells treated with H(2)O(2). An E. coli O157:H7 tpx mutant, with a defect in thioredoxin-dependent thiol peroxidase (Tpx), was more severely damaged by EGCG when compared with its wild type. Similar differing effects were observed in other Gram-positive and Gram-negative bacteria when exposed to EGCG; it caused aggregated in Streptococcus mutans, while it caused grooves in Pseudomonas aeruginosa. AFM results suggest that the major morphological changes of Gram-negative bacterial cell walls induced by EGCG depend on H(2)O(2) release. This is not the case for Gram-positive bacteria. Oxidative stress in Gram-negative bacteria induced by EGCG was confirmed by flow cytometry.

Heat and lime-treatment as effective control methods for E. coli O157:H7 in organic wastes.

Land-application of abattoir wastes is economically appealing and may provide an effective means of closing the nutrient cycling loop. This practise is constrained, however, by legislation which necessitates pre-treatment to remove pathogenic micro-organisms prior to land-spreading. Here we investigated whether heat-treatment or lime addition could eliminate Escherichia coli O157:H7 from three contrasting abattoir wastes. We found that treatment at 60 degrees C for 10 min effectively eradicated the organism while treatment for the same length of time at 50 degrees C led to 2-4 log reductions, but not a complete kill. Temperatures of 72 degrees C induced waste solidification rendering its use impractical. The potential for re-growth in heat-treated and untreated wastes was also investigated. Survival was significantly greater in heat-treated wastes, although the difference was less than half a log unit in magnitude. This effect of heat-treatment on pathogen survival appeared to be ameliorated when wastes were mixed with soil. No viable E. coli O157:H7 cells were recovered from any waste after application of lime (CaO) at a rate of 10 gl(-1), even after enrichment. Our results indicate that pasteurisation-style or liming treatments may provide a suitable alternative method for reducing pathogen loads in abattoir wastes, so that they can be applied to land with minimal biological risk.