Abstract
Escherichia coli O157:H7 (O157) causes human diarrheal disease and healthy cattle are its primary reservoir. O157 colonize the bovine epithelial mucosa at the recto-anal junction (RAJ). Previous studies show that O157 at this site are not eliminated by aggressive interventions including applications of O157-specific lytic bacteriophages and other bactericidal agents. We hypothesize that some O157 at the RAJ mucosa are protected from these killing agents by host cell internalization. To test this hypothesis, rectal biopsies from O157 culture positive and negative cattle were analyzed by fluorescent microscopy and subjected to gentamicin protection assays. GFP-labeled bacteria were found located deep within the tissue crypts and a small number of O157 were recovered from rectal biopsies after gentamicin treatment. Primary bovine rectal epithelial (PBRE) cell cultures were incubated with O157 and subjected to gentamicin protection assays. Strains ATCC 43895, 43894, Sakai, and WSU180 entered the PBRE cells with different levels of efficiency ranging from 0.18 to 19.38% of the inocula. Intracellular bacteria were confirmed to be within membrane-bounded vacuoles by electron microscopy. Cytochalasin D curtailed internalization of O157 indicating internalization was dependent on eukaryotic microfilament assembly. Strain ATCC 43895 exhibited the highest efficiency of internalization and survived for at least 24 h within PBRE cells. Deletion mutation of intimin or its receptor in ATCC 43895 did not reduce bacterial internalization. This strain produced more biofilm than the others tested. Retrospective analysis of cattle challenged with two O157 strains, showed ATCC 43895, the most efficient at host cell internalization, was most persistent.
Highlights
Enterohemorrhagic Escherichia coli (EHEC) cause human disease ranging from self-limited watery diarrhea to the life-threatening condition of hemorrhagic colitis and its sequelae – the hemolytic uremic syndrome (HUS; Karmali et al, 1983; Paton and Paton, 1998)
E. coli O157:H7 in biopsy tissue survived gentamicin treatment and was deep in the mucosal crypts To determine if E. coli O157:H7 were internalized by bovine epithelial cells at the terminal rectum, rectal tissue biopsies from culture positive cattle were analyzed using a gentamicin protection assay
To test whether intimin or translocated intimin receptor (Tir) had a role in internalization by the Primary bovine rectal epithelial (PBRE) cells, we examined the ability of intimin or Tir deletion mutants of both 43894 and 43895 to enter the PBRE cells
Summary
Enterohemorrhagic Escherichia coli (EHEC) cause human disease ranging from self-limited watery diarrhea to the life-threatening condition of hemorrhagic colitis and its sequelae – the hemolytic uremic syndrome (HUS; Karmali et al, 1983; Paton and Paton, 1998). Because the terminal rectal mucosa may be the ideal target for controlling E. coli O157:H7 in cattle, we and others have applied O157-specific lytic bacteriophages (Sheng et al, 2006a; Rivas et al, 2010) or chemical chlorhexidine (Naylor et al, 2007) directly to this location. When these treatments are effective they reduce the level of the E. coli O157:H7 carriage, but never completely eliminate E. coli O157:H7 from the rectal mucosa
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