Cronobacter Support
05-18-2009, 03:06 PM
1st International Conference on Cronobacter Poster Abstract 36
Pathogenesis of Cronobacter: enterotoxin production, adherence and invasion of the blood-brain barrier.Although Cronobacter sakazakii has been implicated in outbreaks causing meningitis and enteritis, we do not know what factor(s) play a role in the transfer of the organism across the blood-brain-barrier in humans, or whether those factors are present in all strains of C. sakazakii. This study assessed whether strains from clinical, food, and environmental sources differed in their ability to adhere and/or invade human brain microvascular endothelial cells. Adhesion and invasion of 30 Cronobacter isolates (10 each from clinical, environmental, and food sources) to human blood-brain-barrier cells was done using a modified gentamicin protection assay. A transposon mutant library was screened in the same manner to identify isogenic mutants showing increased and/or decreased adherence and invasion. The 30 strains were tested for enterotoxin production in vitro using a Vero cell assay. All strains adhered to endothelial cells, and all but 2 clinical strains were able to invade. Interestingly, 70% of clinical strains were positive or indeterminate for capsule production, as compared to 40% and 30% for food and environmental isolates, respectively. Enterotoxin production varied amongst the 30 strains tested. Of the 6 most virulent strains, 5 were of food and one was of environmental origin. SDS-PAGE revealed a distinct protein band present at 66 kDa, the reported molecular weight of the enterotoxin. N-terminal sequencing was done on the excised protein. There did not appear to be any direct correlation between the source of Cronobacter strains and their adherent or invasive abilities. Investigation into the transposon insertion sites in the genomes of non-adherent and non-invasive mutants are underway and should help shed some light on the identity of the factor(s). Capsule formation may be important in the blood-brain barrier pathogenesis.
Franco Pagotto, Jeffrey M. Farber, Graeme Ferguson and Amy Louise Commodore
Health Canada, Bureau of Microbial Hazards, Food Directorate, Ottawa, Ontario, Canada.
Pathogenesis of Cronobacter: enterotoxin production, adherence and invasion of the blood-brain barrier.Although Cronobacter sakazakii has been implicated in outbreaks causing meningitis and enteritis, we do not know what factor(s) play a role in the transfer of the organism across the blood-brain-barrier in humans, or whether those factors are present in all strains of C. sakazakii. This study assessed whether strains from clinical, food, and environmental sources differed in their ability to adhere and/or invade human brain microvascular endothelial cells. Adhesion and invasion of 30 Cronobacter isolates (10 each from clinical, environmental, and food sources) to human blood-brain-barrier cells was done using a modified gentamicin protection assay. A transposon mutant library was screened in the same manner to identify isogenic mutants showing increased and/or decreased adherence and invasion. The 30 strains were tested for enterotoxin production in vitro using a Vero cell assay. All strains adhered to endothelial cells, and all but 2 clinical strains were able to invade. Interestingly, 70% of clinical strains were positive or indeterminate for capsule production, as compared to 40% and 30% for food and environmental isolates, respectively. Enterotoxin production varied amongst the 30 strains tested. Of the 6 most virulent strains, 5 were of food and one was of environmental origin. SDS-PAGE revealed a distinct protein band present at 66 kDa, the reported molecular weight of the enterotoxin. N-terminal sequencing was done on the excised protein. There did not appear to be any direct correlation between the source of Cronobacter strains and their adherent or invasive abilities. Investigation into the transposon insertion sites in the genomes of non-adherent and non-invasive mutants are underway and should help shed some light on the identity of the factor(s). Capsule formation may be important in the blood-brain barrier pathogenesis.
Franco Pagotto, Jeffrey M. Farber, Graeme Ferguson and Amy Louise Commodore
Health Canada, Bureau of Microbial Hazards, Food Directorate, Ottawa, Ontario, Canada.