Identical Penicillin-Binding Domains in Penicillin-Binding Proteins of Streptococcus pneumoniae Clinical Isolates with Different Levels of β-Lactam Resistance

Abstract

We have sequenced the penicillin-binding domains of the complete repertoire of penicillin-binding proteins and MurM from 22 clinical isolates of Streptococcus pneumoniae that span a wide range of ␤-lactam resistance levels. Evidence of mosaicism was found in the genes encoding PBP 1a, PBP 2b, PBP 2x, MurM, and, possibly, PBP 2a. Five isolates were found to have identical PBP and MurM sequences, even though the MICs for penicillin G ranged from 0.25 to 2.0 mg/liter. When the sequences encoding PBP 1a, PBP 2b, and PBP 2x from one of these isolates were used to transform laboratory strain R6, the resulting strain had a resistance level higher than that of the less resistant isolates carrying that PBP set but lower than that of the most resistant isolates carrying that PBP set. This result demonstrates that if the R6 strain is arbitrarily defined as the standard genotype, some wild genetic backgrounds can either increase or decrease the PBP-based resistance phenotype.The mounting rates of resistance of Streptococcus pneumoniae to antibiotics is an important concern, as this human pathogen is responsible for serious diseases, such as otitis, pneumonia, meningitis, and bacteremia. In clinical isolates, resistance to ␤-lactams is mostly due to the expression of modified penicillin-binding proteins (PBPs), the targets of the ␤-lactams, with reduced affinities for the antibiotics (3). The low-affinity PBPs are the products of mosaic genes that result from multiple events of homologous recombination with genes from other strains or closely related species (3). The presence of a functional murM gene (also called fibA), which is involved in the synthesis of an alternative physiological substrate for the PBPs, is required for the expression of ␤-lactam resistance (9, 31). Mosaic murM genes can dramatically increase the level of resistance due to low-affinity PBPs (9, 28).S. pneumoniae has five high-molecular-weight PBPs, three of which (PBP 2x, PBP 2b, and PBP 1a) are definitely involved in ␤-lactam resistance (23), and one low-molecular-weight PBP. A laboratory experiment showed that the five high-molecularweight PBPs were modified upon transfer of high-level resistance from Streptococcus mitis to S. pneumoniae (13). Other works have suggested a role for PBP 2a (26,27) and the low-molecular-weight PBP, PBP 3 (17), in ␤-lactam resistance. These findings raised the possibility that previously undetected variability in all the pbp genes may account for the wide range of levels of resistance to ␤-lactams.On the basis of the premise that knowledge of the sequence of MurM and all the PBPs might be sufficient to predict the level of ␤-lactam resistance, we sequenced murM and the region coding the penicillin-binding domain of the six PBPs from 22 isolates of S. pneumoniae. Five isolates were found to have very different MICs for ␤-lactams, even though they had the same six penicillin-binding domains and MurM. This demonstrates that other genes modulate significantly the resistance provided by the low-affinity PBPs. By transferring ...

First Characterization of Heterogeneous Resistance to Imipenem in Invasive Nontypeable Haemophilus influenzae Isolates

“…Isolates of Streptococcus pneumoniae carrying identical PBP sequences can show different levels of ␤-lactam resistance (6). Moreover, the transfer of the pbp genes from a clinical isolate to the laboratory strain R6 resulted in a transformant with a higher level of resistance than that of the donor strain (6). Possibly, genetic factors can either increase or decrease the level of PBP-based resistance in relation to the compatibility of the mutated PBP with other enzymes involved in peptidoglycan synthesis (6).…”

“…We speculated that the genetic background of strain Rd may modulate the level of resistance to imipenem conferred by PBP 3 on the transformant. Isolates of Streptococcus pneumoniae carrying identical PBP sequences can show different levels of ␤-lactam resistance (6). Moreover, the transfer of the pbp genes from a clinical isolate to the laboratory strain R6 resulted in a transformant with a higher level of resistance than that of the donor strain (6).…”

“…Moreover, the transfer of the pbp genes from a clinical isolate to the laboratory strain R6 resulted in a transformant with a higher level of resistance than that of the donor strain (6). Possibly, genetic factors can either increase or decrease the level of PBP-based resistance in relation to the compatibility of the mutated PBP with other enzymes involved in peptidoglycan synthesis (6). However, S. pneumoniae is homogeneously resistant to ␤-lactam antibiotics, which means that each bacterial cell has the same degree of resistance, while the NTHI isolates described herein, including the Rd/183 transformant, were heterogeneously resistant to imipenem.…”

Inactivation Kinetics of a New Target of β-Lactam Antibiotics

“…Only two of the seven steps led to substitutions in Ldt fm , indicating that modifications of other unknown factors are involved in acquisition of resistance. For the DD-transpeptidases, emergence of ␤-lactam resistance also involves a combination of multiple modifications affecting non-penicillin-binding protein factors, several residues of a given penicillin-binding protein, and sometimes several penicillin-binding proteins (11)(12)(13)(14)(15)(16). The requirement for multiple mutations accounts for the relative robustness of ␤-lactams with respect to the emergence of resistance by target modification.…”

Biological and Epidemiological Features of Antibiotic-Resistant Streptococcus pneumoniae in Pre- and Post-Conjugate Vaccine Eras: a United States Perspective

“…The scenario that different PBP allele combinations confer differing ␤-lactam resistance phenotypes is complicated by the finding that transformations using PBP genes from certain strains were not sufficient to transform wild-type strains to the same high level of resistance (120,121). One study reported that strains exhibiting identical PBP transpeptidase domain sequences exhibited penicillin MICs ranging from 0.25 to 2.0 g/ml (122).…”