Rapid screening for penicillin susceptibility of systemic pneumococcal isolates by restriction enzyme profiling of the pbp2B gene

Abstract

Restriction digest profiling of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. The pbp2b amplicon of all pneumococcal isolates for which the MICs of penicillin were < or = 0.03 microgram/ml had one of two different susceptible restriction profiles, and all 33 isolates for which MICs were 0.5 microgram/ml or greater had one of seven distinct resistant profiles. Low-concentration penicillin resistance (MICs = 0.06 microgram/ml to 0.25 microgram/ml) was associated with sensitive HaeIII profiles in some isolates; however, RsaI profiling and pbp2b sequence analysis of such isolates revealed that some isolates contained low-level resistant pbp2b alleles, while others had susceptible pbp2b alleles. This data indicates that low-level penicillin resistance is sometimes conferred by determinants other than pbp2b.

FIG. 1

HaeIII profiles of pbp2b amplicons from penicillin-susceptible (MICs, ≤0.03 μg/ml) (s1 and s2) and penicillin-resistant (MICs, 0.06 to 4.0 μg/ml) (r1 to r9) clinical blood isolates of pneumococci. Lane 1, r2; lane 2, r8; lane 3, r5; lane 4, r4; lane 5, r3; lane 6, r1; lane 7, r9; lane 8, r7; lane 9, r6; lane 10, s2; lane 11, s1, lane 12, Gibco-BRL kilobase ladder.

FIG. 2

Comparison of the pbp2b sequence of low-level penicillin-resistant (MIC, 0.25) pneumococcal isolate 5 with that of penicillin-susceptible strain R6. Nucleotide numbering was taken from reference . Codons encoding amino acid substitutions and the indicated RsaI site are in bold-faced type. Nonconservative codon substitutions and a perfect match to the R2 primer sequence used by du Plessis et al. in detecting penicillin resistance (5a) are underlined.

FIG. 3

pbp2b amplicon RsaI profiles of pneumococci susceptible to penicillin and of pneumococci with low-level resistance. Lanes 1, 5, and 8 are profiles of isolates for which MICs were 0.25 μg/ml; lanes 1 and 8 are from isolates 11 and 5, respectively. Lanes 6 and 7 are profiles of isolates for which MICs were 0.12 μg/ml. Lanes 2 to 4 and 9 to 10 are profiles of penicillin-susceptible isolates (MICs, ≤0.03 μg/ml). Lane 11, Gibco-BRL 1-kb ladder.