In vitro selection of one-step mutants of Streptococcus pneumoniae resistant to different oral beta-lactam antibiotics is associated with alterations of PBP2x

Abstract

Many oral penicillins and cephalosporins are used to treat clinical infections caused by Streptococcus pneumoniae. Therefore, using different beta-lactams as selectors, we estimated the frequencies of one-step mutations leading to resistance. Resistant mutants were obtained from penicillin-susceptible, intermediately resistant, and penicillin resistant strains. For cefixime, cefuroxime, cefpodoxime, cefotaxime, and ceftriaxone, the frequencies of mutation ranged from 10(-6) to 10(-8) when resistant mutants were selected at 2- to 8-fold the MIC, and the MICs increased 2- to 16-fold. For ampicillin, ampicillin-sulbactam, amoxicillin, amoxicillin-clavulanic acid, cefaclor, and loracarbef, the frequencies of mutation were about 10(-7) to 10(-8), and the MICs increased twofold at most. One to three resistance profiles of the resulting mutants were selected for each of the selecting antibiotics. Among those, some showed resistance to the cephalosporins associated with a 2- to 32-fold increase in susceptibility to the penicillins. Competition experiments showed a decreased affinity of PBP2x for cefpodoxime in all mutants. In some mutants that were more susceptible to amoxicillin, a decreased affinity of PBP2x for cefpodoxime was associated with an increased affinity for amoxicillin and a particular substitution of alanine for threonine at position 550 just after the KSG triad. From these results we infer (i) that among the beta-lactams tested the penicillins, cefaclor, and loracarbef selected one-step resistant mutants less frequently and that they achieved a lower level of resistance, and (ii) that mutants with different profiles may have acquired different point mutations in PBP2x.

Resistant penicillin-binding proteins

“…1). Two of these sites known to contribute to resistance are shown by the substitution Thr338 to Ala or to Pro immediately after the active site Ser337 (J. Krauß and R. Hakenbeck, unpublished results), and by the Gln552 to Glu mutation [39]. Furthermore, the His394 to Tyr mutation was identified in a cefotaxime-resistant mutant [22] and occurs also in a PBP2x of a resistant S. pneumoniae.…”

Penicillin-binding Protein 2x of Streptococcus pneumoniae: Three New Mutational Pathways for Remodelling an Essential Enzyme into a Resistance Determinant

“…10 S. pneumoniae M31 containing the Q552E mutation in PBP2x has been described and was kindly provided by Laurent Gutmann. 13 S. pneumoniae strains were grown in liquid culture in a casein-based semisynthetic medium supplemented with 0.2% yeast extract. 41 MICs of beta-lactam antibiotics were tested by agar dilution on blood agar plates (3% sheep blood).…”

“…This was not surprising for T550A and Q552E, mutations that have been shown to be selected easily with cefotaxime in the laboratory. 13,18,19 Considering the group I PBP2x, mutations located between G597 and G601 also decreased the cefotaxime susceptibility in the R6 background. The variability of mutations that are located at the N-terminal end of α11 (G597D, L600W and G601V) strongly suggests that this region is critical for the active-site conformation.…”

“…1c). These include Q552E, which has been shown to contribute to cephalosporin resistance, 9,13,28 and S389L, suggested to contribute to a destabilizing effect of the active site. 17 In addition, there are three unusual alterations not found in other clinical isolates including major resistant clones: G286P, F388L and A604K.…”

“…[10][11][12][13][14][15][16][17] PBP2x mutations selected in a set of independently obtained spontaneous cefotaxime-resistant laboratory mutants revealed a surprising multitude of sites involved in resistance development for this essential protein, all of which were scattered throughout the penicillinbinding domain. 10,11 Mutations relevant for resistance in the mosaic genes of clinical isolates appear to be distinct, and only the mutation T550A, which can easily be selected in the laboratory, 18,19 has been found occasionally in high-level cefotaxime-resistant isolates 12,20 and as a single mutation in lowlevel resistant strains.…”