Identification of Penicillin and other Beta-Lactam Resistance in Streptococcus Pneumoniae by Polymerase Chain Reaction

Abstract

To identify penicillin (Pc) and other β-lactam resistance in 310 clinical isolates of Streptococcus pneumoniae by polymerase chain reaction (PCR), 3 sets of primers were designed to amplify Pc-binding protein (PBP) genes previously detected in Pc-susceptible strains: 1) a 430-bp fragment of the pbp1a gene, 2) a 292-bp fragment of the pbp2x gene, and 3) a 77-bp fragment of the pbp2b gene. The amplified regions of each PBP gene were positioned in highly divergent sequences of Pc-resistant S. pneumoniae. In other words, isolates for which these DNA fragments were detected were regarded as possessing sequences almost the same as that of the susceptible R6 strain and those for which these DNA fragments were not detected were assumed to have mutations. A set of primers that amplify 273 bp of the autolysin (lytA) gene to identify S. pneumoniae was applied as well. Of 166 isolates for which the minimum inhibitory concentration (MIC) of Pc were ≤0.06μg/mL, 83 (50.0%) were confirmed to be true susceptible strains with no PBP gene mutation and most of the remaining strains were found to possess pbp2x mutation. In contrast, most of 109 isolates for which the MIC of Pc were ≥0.5 μg/mL were confirmed to possess mutations in all three PBP genes. Thirty-five strain for which the MIC of Pc ranged from 0.125 to 0.25μg/mL possessed various PBP gene mutations. The relationships between susceptibilities to 9 β-lactams of S. pneumoniae and PBP gene mutation were analyzed by multiple regression analysis. Antibiotics were classified into 4 types according to the differences in PBP gene mutation affecting their MIC levels, 1) the MIC of Pc and ampicillin were affected by pbp1a and pbp2b mutations; 2) those of cefotaxime, cefpodoxime, and cefditoren were affected clearly by pbp2x mutation; 3) those of cefaclor and cefdinir were affected more strongly by pbp1a mutation than the pbp2x; and 4) the MIC of faropenem and imipenem were affected strongly by pbp2b mutation. These findings suggest that it may be possible to easily determine whether a S. pneumoniae isolate is susceptible or resistant to Pc, cefotaxime, and other β-lactams by applying PCR using a combination of primers.