Additionally, two clusters (6B and check details 12) suggested genetic relationship (by three band difference) between isolates assigned to phylogroups (eBURST group 2 and Clade 13, respectively) and isolates with no phylogroup assignment, probably reflecting distant phylogenetic relationship not Mocetinostat mw detected by the parsimony analysis. Phylogeny and resistance genotypes The 116

rPBP3 and 80 sPBP3 isolates were distributed on 32 and 44 STs, respectively. Six of the 70 STs in this study (ST12, ST57, ST155, ST159, ST411 and ST422) included both categories. Most rPBP3 isolates (102/116, 88%) belonged to five phylogroups (rPBP3 proportions in brackets): eBURST group 2 (45/50, 90%); Clade 13 (28/59, 47%); Clade 9 (22/26, 85%); Clade 8 (5/8, 63%) or Clade 10 (2/4, 50%). The remaining 14 rPBP3 isolates lacked phylogroup assignment. The two group III-like and the single

group III high-rPBP3 isolates were ST160 (no phylogroup) and ST1197 (Clade 13), respectively. No isolates in Clade 1 (n = 5), Clade 2 (n = 4), Clade 6 (n = 1), Clade 11 (n = 5) and Clade 12 (n = 2) were rPBP3. The ftsI alleles lambda-2, zeta and omicron, BMS202 datasheet encoding the three most frequent PBP3 types A, B and D, respectively, were, with a few notable exceptions, carried by ST367 (eBURST group 2), ST396 (Clade 9) and ST201 (Clade 13) (Figure 3). In addition, PBP3 type A encoded by the slightly different allele lambda-1 was present in ST14, a triple locus variant of ST367 (both STs belong to eBURST group 2). These four strains (defined by combinations of STs and ftsI alleles) accounted for 61% (71/116) of the rPBP3 isolates in the current study. Two strains frequently occurring in this study (ST14 with PBP3 type A and ST396 with PBP3 type B) had PFGE band patterns and ftsI alleles identical to strains in the two most prevalent resistant clones three years earlier (PFGE clusters 1 and 2, respectively) (Figure 4) [11]. Apart from ST367, PBP3 type A encoded by lambda-2 was present in the following unrelated STs: ST57 (Clade 8), ST85 (Clade 9) and ST12 (no phylogroup). Similarly, the ftsI allele gamma, encoding

PBP3 type H, was present in ST12 (no phylogroup) as well as the unrelated ST411 and ST422 (Clade (-)-p-Bromotetramisole Oxalate 10). Conversely, seven STs hosted more than one PBP3 type. Notably, the six ST57 isolates carried four highly divergent rPBP3 types (A, K, L and N) and the reference sequence (z0). Three ST57 isolates were TEM-1 positive but only one isolate had both TEM-1 and rPBP3. Most isolates with both resistance mechanisms (5/7, 71%) were ST396. Clinical characteristics Clinical information for the 196 study isolates and the 599 remaining isolates in the original population is summarized in Table 4. For the study isolates, median age and age range of the patients were 5 (0 – 86) yrs with a male/female ratio of 1.0. The corresponding numbers in the original population were 5 (0 – 97) and 1.0.