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°C were inoculated with an E. coli O157:H7 cell suspension at 2 or 25°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 m below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 m. 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.

Wastewater reuse in irrigation: A microbiological perspective on implications in soil fertility and human and environmental health

“…Moreover, because plants are privileged hosts for these bacteria, if supplied in irrigation water, they can proliferate and survive for longer periods when plants are present than in their absence (Gagliardi and Karns, 2002;Ibekwe et al, 2004;Tyler and Triplett, 2008). Another risk is that bacteria transmitted by this pathway may be inherited through plant seeds, as has been described for some endophytic bacteria (Burnett et al, 2000;Cooley et al, 2003;Guo et al, 2001;Rosenblueth and Martínez-Romero, 2006;Tyler and Triplett, 2008;Wang et al, 2006). The risks associated with the dis-semination of human pathogens due to the use of treated wastewater for irrigation can be considered low (Bichai et al, 2012;Shuval and Fattal, 2003).…”

“…Although the risks of propagation of phytopathogens to soils through wastewater are scarcely addressed in literature, it is recognised that several plant pathogens, either bacteria, fungi, viruses, parasitic nematodes or oomycetes (e.g. Phytophthora and Pythium) are waterborne (Bush et al, 2003;Hong and Moorman, 2005). Evidences of possible risks are supported by reports involving the use of recycled freshwater in plant nurseries and greenhouses, concluding that this can be identified as an important source and vehicle for the spread of plant pathogens (Stewart-Wade, 2011).…”

Mass and Density Measurements of Live and Dead Gram-Negative and Gram-Positive Bacterial Populations

“…Escherichia coli O157:H7, ATCC 43888, was grown in Luria-Bertani (LB) broth overnight to stationary phase (10 8 to 10 9 cells/ml). E. coli experiments were carried out with the minimal salt medium (MSM) described by Burnett et al designed specifically for E. coli O157:H7 (22). Listeria innocua ATCC 33090 was grown in brain heart infusion (BHI) broth overnight to stationary phase (10 8 to 10 9 cells/ml).…”

Kinetics and Strain Specificity of Rhizosphere and Endophytic Colonization by Enteric Bacteria on Seedlings of Medicago sativa and Medicago truncatula

“…Various approaches to surface sterilize vegetables do not eliminate the enteric pathogens (2,24,25,26). This result led to the discovery that these enteric pathogens reside within the interiors of apples, lettuce, and alfalfa sprouts (4,11,16,26). Gandhi et al (11) showed that a Salmonella Stanley strain could reside within the interiors of alfalfa sprouts to a depth of 18 m from the surface.…”