COMBINATIONAL DETECTION OF AUTOLYSIN AND PENICILLIN-BINDING PROTEIN 2B GENES OF STREPTOCOCCUS-PNEUMONIAE BY PCR

PCR was used to identify penicillin resistance in 1,062 clinical isola tes of Streptococcus pneumoniae. Three sets of primers were designed t o amplify (i) a 240-bp fragment of the penicillin-binding protein (PBP ) 2B gene (pbp2b) of penicillin-susceptible S. pneumoniae (PSSP), (ii) a 215-bp fragment of the class A mutations of the pbp2b gene present in penicillin-resistant S, pneumoniae, and (iii) a 286-bp fragment of the class B mutation, In addition, a set of primers that amplify 273 b p of the autolysin (lytA) gene was applied in combination with the abo ve to identify S. pneumoniae. Of 621 isolates for which MICs of penici llin were less than or equal to 0.06 mu g/ml, 614 (98.9%) were ascerta ined as having DNA fragments amplified by the PSSP primers. Of 441 iso lates for which MICs of penicillin were greater than or equal to 0.125 mu g/ml, a class A mutation was detected in only 8 (1.8%), a class B mutation was detected in 310 (70.3%), and neither class A nor class B mutations were found in the remaining 123 (27.9%). However, when analy sis was limited to isolates for which MICs of penicillin were greater than or equal to 1.0 mu g/ml, 247 isolates (89.8%) of 275 were found t o possess a class B mutation. When PBPs were analyzed in 12 isolates w ith unclear mutations of the pbp2b gene by using [H-3]benzylpenicillin , low affinity to PBP 2B was observed in them all. These findings sugg est that a pbp2b mutation other than class A or class B is present in these isolates. These results also indicate that it may be possible to identify PSSP and penicillin-resistant S. pneumoniae by applying PCR using a combination of primers to detect the susceptible pbp2b gene, r esistant pbp2b gene mutations. and the IytA gene.