P-063. Phenotypic Microarray of Escherichia coli O157:H7 Isolates from the 2006 Outbreaks Linked to Fresh Produce

A. Mukherjee, M. K. Mammel, J. E. LeClerc, T. A. Cebula;
US. FDA, Laurel, MD.

Background: In order to identify phenotypic markers to track foodborne pathogens, phenotypic microarray (PM) was conducted on Escherichia coli O157:H7 isolates involved in the three outbreaks of 2006 linked to fresh produce. We have recently shown that 98% of E. coli O157:H7 from a reference collection of 120 isolates can utilize N-acetyl-D-galactosamine (Aga), but none of 194 isolates of E. coli O157:H7 from the 2006 spinach outbreak did (J. Bacteriol. 2007, doi 10.1128/JB. 01737-07). Here we have extended the PM studies on outbreak isolates of E. coli O157:H7 linked to lettuce in Taco Bell (L1) and Taco John (L2) restaurants and compare their phenotypic properties. Another phenotype of E. coli O157:H7 is that it utilizes sucrose but not D-serine because it has the sucrose regulon integrated into the D-serine operon. We therefore tested all isolates from the three fresh produce outbreaks for sucrose and D-serine utilization and identified the genetic basis in three isolates from the spinach outbreak that renders them unable to utilize sucrose. Methods: The Biolog system was used for PM studies of E. coli O157:H7 isolates. Genome sequencing of one of the spinach isolates that could not utilize sucrose was done by the 454 method. Pyrosequencing was done to confirm the mutation in the other two isolates that did not utilize sucrose. Results: PM of 20 isolates linked to lettuce from L1 and 12 isolates linked to lettuce from L2 showed that the L1 isolates did not utilize Aga whereas the L2 isolates utilized Aga. Screening a total of 226 isolates associated with the three outbreaks showed that all but three isolates from the spinach outbreak utilized sucrose. Whole genome sequencing of one of the three isolates showed that a point mutation in z3623 gene, the sucrose transporter, results in a premature stop codon (Trp26; TGG to TAG). Pyrosequencing showed that all three isolates had the same mutation. Conclusion: The Aga phenotype studies showed that the spinach and L1 isolates are related but the L2 isolates are distinctly different. There are three variants among the spinach isolates with respect to sucrose utilization. These results show the usefulness of metabolic profiling in molecular epidemiology investigations.