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coding regions of the transpeptidase domain of the pbp1a, pbp2b and pbp2x genes of the two GBS isolates were amplified and sequenced according to previously outlined methods. 4 Amino acid sequences were deduced and analysed using the ClustalW alignment tool included in the Lasergene software (DNAstar, Madison, WI, USA). Nucleotide and deduced amino acid sequences were compared with those of the reference penicillin-susceptible strains 2603V/R (GenBank accession number: NC_004116) and NEM316 (GenBank accession number: NC_004368). DNA analysis revealed that the pbp genes of the clinical GBS isolates possessed many amino acid substitutions compared with the corresponding genes of the reference strains 2603V/R and NEM316. Five previously described amino acid substitutions were observed in both the penicillin-susceptible GBS and penicillin G-non-susceptible GBS isolates (S453N and N682D in PBP1a, V625I in PBP2b, and I377V and G627V in PBP2x). 4 However, there were three novel substitutions (T526A in PBP1a, P278L in PBP2b and N575D in PBP2x) found exclusively in the penicillin G-non-susceptible GBS 2007 isolate. In previous studies, the V405A and Q557E substitutions adjacent to the conserved SSN and KSG motifs in PBP2x, considered to form the active site of the enzyme, were found in 4 invasive GBS with elevated, but still susceptible, MICs of one or multiple b-lactam antibiotics, in 21 penicillin G-non-susceptible GBS isolated from the respiratory tract and in a penicillin G-non-susceptible GBS recurrently isolated from a sacral ulcer. 2 – 5 Several amino acid substitutions in PBP1a, PBP2a and PBP2b were also found in penicillin G-non-susceptible GBS isolated from the respiratory tract. 4 However, the PBP2a amino acid substitutions were documented in only two GBS with penicillin MICs of 1 mg/L. 4 In the present study, the penicillin G-non-susceptible GBS 2007 isolate did not harbour the PBP2x V405A and Q557E substitutions previously associated with reduced susceptibility to penicillin. 2 – 5 Instead, the penicillin G-non-susceptible GBS 2007 isolate possessed three novel substitutions (T526A in PBP1a, P278L in PBP2b and N575D in PBP2x). At this time it is not known whether these substitutions can actually increase resistance to penicillin since they were not found within or in the proximity of the putative conserved motifs, and their significance needs to be assessed in future studies. Moreover, it is possible that the penicillin G-non-susceptible GBS 2007 isolate harboured mutations in other pbp or non-pbp genes that are responsible for the observed phenotype. To our knowledge, that is the first report of development …

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