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

Confocal scanning laser microscopy (CSLM) was used to demonstrate the attac hment of Escherichia coli O157:H7 transformed with a plasmid encoding for g reen fluorescent protein (GFP) to the surface and within the internal struc tures of nonwaxed Red Delicious cv. apples. Apples at 2 or 25 degreesC were inoculated with an E. coil O157:H7 cell suspension at 2 or 25 degreesC, Th e 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 (a tmospheric versus 10,130 Pa), on the attachment and infiltration of cells w as determined. CSLM stereo images of external surfaces of apples subjected to all combinations of test parameters showed preferential cellular attachm ent to discontinuities in the waxy cuticle on the surface and to damaged ti ssue surrounding puncture wounds, where the pathogen was observed at depths up to 70 mum below the skin surface. Attachment to lenticels was sporadic but was occasionally observed at depths of up to 40 mum, Infiltration throu gh the floral tube and attachment to seeds, cartilaginous pericarp, and int ernal trichomes were observed in all apples examined, regardless of tempera ture differential during inoculation. The pressure differential had no effe ct on infiltration or attachment of E. coil O157:H7, Image analysis to coun t cells at various depths within tissues was used to quantitatively compare the extent of infiltration into various apple structures as well as the ef fects of the temperature differential. Puncture wounds harbored greater num bers of the pathogen at greater depths than did other sites examined. Attac hment or infiltration of cells was greater on the intact skin and in lentic els, russet areas, and the floral tube of apples inoculated under a negativ e temperature differential compared to those inoculated under no temperatur e differential, The results suggest that E. call O157:H7 attached to intern al core structures or within tissues of apples may evade decontamination tr eatments. Interventions designed to deliver disinfectants to these location s or to remove viable cells off. coil O157:H7 and other pathogens from appl es by other means need to be developed and validated.