Objectives: To phenotypically and genotypically characterize 11 strains (isolated in four different centres) exhibiting penicillin MIC of 8-32 mg/L among isolates of the SPICE project. Nine isolates were from Romania (9/162; 5.56%) and two from Poland (2/305; 0.66%).
Methods: In vitro susceptibility was determined in triplicate by microdilution (CLSI guidelines), and additionally, MICs of penicillin, cefotaxime and amoxicillin were confirmed in triplicate by agar dilution. Multilocus sequence typing (MLST), PFGE and gene amplification and sequencing were performed.
Results: For the nine Romanian isolates, MICs were >/=16 mg/L for penicillin, cefotaxime and amoxicillin, >/=32 mg/L for cefuroxime and cefpodoxime, 4-8 mg/L for cefditoren and >/=128 mg/L for erythromycin and gentamicin. All isolates were non-susceptible to imipenem (MIC = 0.5-1 mg/L) and susceptible to levofloxacin (MIC = 0.5-1 mg/L) and vancomycin (MIC = 0.25-0.5 mg/L). These Romanian strains presented a new cluster in the 595-600 region of PBP2X (YSGIQL-->LSTPWF) conferring 98% homology with Streptococcus mitis PBP2X, with a new MurM allele (seven strains) with eight amino acid changes versus R6. PBP nucleotide sequences were highly conserved suggesting a common origin. Allelic profiles of two strains gave sequence type 321, three strains exhibited a single- and four a double-locus variance. MLST-predicted serotype was 23F in all but one strain (19F), but three strains were 19A by Quellung.
Conclusions: The multidrug high resistance (precluding adequate oral therapy in children), its origin, the prevalence found in Romania and the presence of non-vaccine (7-valent) serotypes should worry the medical community because of a possible clonal diffusion that would limit therapeutic alternatives.