Gaps were not considered an extra state. The Jukes-Cantor correction was used to compensate for divergence being a logarithmic function of time due to the increased probability of a second substitution at a nucleotide site slowing the increase in the count of differences as divergence

BVD-523 in vitro time increases [23]. Felsenstein bootstraps (1,000 simulations) were applied to assess the level of confidence for each clade of the observed trees based on the proportion of bootstrap trees showing the same clade [24]. The topology of the maximum parsimony tree was optimized using simulated annealing. [This is a heuristic approach that occasionally accepts a worse tree during the course of the search allowing it to escape local optima. This method is more economical than the more usual heuristic searches (stepwise addition and hill-climbing), which can require many random re-starts, especially with large data matrices]. Figure 1 recN gene sequencing clustering analysis of Cronobacter species (Colours relate

to the phenotypes in Table 3). Results Isolation & Identification A total of sixteen Cronobacter strains were isolated from various food products (Table 1). Some of the non-Cronobacter strains isolated included Citrobacter freundii, Enterobacter cloacae, Proteus Exoribonuclease vulgaris and putative Vibrio cholerae. selleck inhibitor Presumptive positive isolates produced blue-green colonies on DFI agar and were identified as Cronobacter (E. sakazakii)

using ID 32E test strips. Real-time PCR analysis confirmed the detection of Cronobacter isolates. Biochemical tests were performed in order to distinguish the phenotypes of the Cronobacter isolates and contribute to the speciation of the collection of strains. The results of these tests are shown in Table 3. Table 3 Results of pheno- and genotyping of Cronobacter isolates. Isolate Species AMG DUL IND INO MAL rep-PCR PFGE CFS-FSMP 1504 C. sakazakii + – - + – B 7 CFS-FSMP 1505 C. sakazakii + – - + – B 7 CFS-FSMP 1502 C. sakazakii + – - + – B 8 CFS-FSMP 1503 C. sakazakii + – - + – B 8 CFS-FSMP 1506 C. sakazakii + – - + – B 8 CFS-FSMP 1511 C. sakazakii + – - + – C 2 CFS-FSMP 1512 C. sakazakii + – - + – C 2 CFS-FSMP 1515 C. sakazakii + – - + – C 2 CFS-FSMP 1513 C. sakazakii + – - + + C 1 CFS-FSMP 1514 C. sakazakii + – - + + C 1 CFS-FSMP 1501 C. sakazakii + – - + + C 3 CFS-FSMP 1507 C. sakazakii – + + – - B 6 CFS-FSMP 1500 C. malonaticus + – - – + A 4 CFS-FSMP 1508 C. malonaticus + – - – + A 4 CFS-FSMP 1510 C. malonaticus + – - – + A 4 CFS-FSMP 1509 C.