Attachment of Escherichia coli O157:H7 to the surfaces and internal structures of apples as detected by confocal scanning laser microscopy

Abstract

Confocal scanning laser microscopy (CSLM) was used to demonstrate the attachment of Escherichia coli O157:H7 transformed with a plasmid encoding for green fluorescent protein (GFP) to the surface and within the internal structures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25 degrees C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25 degrees C. The effect of a negative temperature differential (cold inoculum, warm apple), a positive differential (warm inoculum, cold apple), and no differential (warm inoculum, warm apple), in combination with a pressure differential (atmospheric versus 10,130 Pa), on the attachment and infiltration of cells was determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachment to discontinuities in the waxy cuticle on the surface and to damaged tissue surrounding puncture wounds, where the pathogen was observed at depths up to 70 microm below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 microm. Infiltration through the floral tube and attachment to seeds, cartilaginous pericarp, and internal trichomes were observed in all apples examined, regardless of temperature differential during inoculation. The pressure differential had no effect on infiltration or attachment of E. coli O157:H7. Image analysis to count cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the effects of the temperature differential. Puncture wounds harbored greater numbers of the pathogen at greater depths than did other sites examined. Attachment or infiltration of cells was greater on the intact skin and in lenticels, russet areas, and the floral tube of apples inoculated under a negative temperature differential compared to those inoculated under no temperature differential. The results suggest that E. coli O157:H7 attached to internal core structures or within tissues of apples may evade decontamination treatments. Interventions designed to deliver disinfectants to these locations or to remove viable cells of E. coli O157:H7 and other pathogens from apples by other means need to be developed and validated.

FIG. 1

Longitudinal cross section of a Red Delicious apple showing the floral tube (a), ventral cavity (b), seed locules (c), and seeds (d).

FIG. 2

CSLM stereo images showing attachment of E. coli O157:H7 on intact apple surface. (A) Cleft (16-μm depth) in the waxy cuticle (open arrow); most cells are attached within the cleft (closed arrow). (B) Clusters of cells (arrow) on intact cuticle 34 μm in height. Cells were inoculated under a negative temperature differential. Bar, 10 μm.

FIG. 3

Infiltration of fluorescent E. coli O157:H7 into external surface structure of apples as affected by negative (○) or positive (□) temperature differentials. The number of cells at various depths below the surface was determined by image thresholding and particle analysis in selected regions (213,000 μm³) of CSLM stacks.

FIG. 4

CSLM stereo images showing attachment of E. coli O157:H7 to apple lenticels and russet areas. (A) Narrow fissure (42-μm depth) radiating from a lenticel heavily colonized with cells (arrow). (B) Lenticel harboring attached (closed arrow) and unattached (open arrow) cells. (C) Unattached and attached cells on wax platelets of russet. Bar, 10 μm.

FIG. 5

CSLM stereo images showing attachment of E. coli O157:H7 to tissue surrounding skin puncture wounds (open arrows). Heavily colonization (closed arrows) of damaged tissue is shown to depths of 48 μm (A) and 70 μm (B) below the surface. Bar, 10 μm.

FIG. 6

CSLM stereo images showing attachment of E. coli O157:H7 to internal trichomes (open arrow) of the floral tube at a depth of 52 μm where cells (closed arrow) are attached to or entrapped within the trichome network (A) and seed integument (18 μm depth), which harbored few attached cells (closed arrow) (B). Bar, 10 μm.

FIG. 7

Infiltration of fluorescent E. coli O157:H7 into the internal structure of apples as affected by negative (○) or positive (□) temperature differentials. The number of cells at various depths below the surface was determined by image thresholding and particle analysis in selected regions (213,000 μm³) of CSLM stacks.

FIG. 8

CSLM stereo images showing attachment of E. coli O157:H7 to the ventral cavity. (A) More attached cells (closed arrow) were observed in crevices (38-μm depth) than on the smooth regions of cartilaginous pericarp (open arrow). (B) Irregular tissue (42-μm depth) on the ventral cavity harboring many cells (closed arrow). Bar, 10 μm.