ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2002, p. 12731280 0066-4804/02/$04.00 0 DOI: 10.1128/AAC.46.5.12731280.

2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 5

Effects of Amino Acid Alterations in Penicillin-Binding Proteins (PBPs) 1a, 2b, and 2x on PBP Afnities of Penicillin, Ampicillin, Amoxicillin, Cefditoren, Cefuroxime, Cefprozil, and Cefaclor in 18 Clinical Isolates of Penicillin-Susceptible, -Intermediate, and -Resistant Pneumococci
Kensuke Nagai,1 Todd A. Davies,1 Michael R. Jacobs,2 and Peter C. Appelbaum1*
Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania,1 and Department of Pathology, Case Western Reserve University, Cleveland, Ohio2
Received 15 October 2001/Returned for modication 7 January 2002/Accepted 24 January 2002

Amino acid alterations in or anking conserved motifs making up the active binding sites of penicillinbinding proteins (PBPs) 1a, 2b, and 2x of pneumococci were correlated with changes in afnities of penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor for these PBPs. Four penicillin-susceptible (PSSP), eight penicillin-intermediate (PISP), and six penicillin-resistant (PRSP) pneumococci were studied by DNA sequencing of the penicillin-binding sites of the pbp1a, -2x, and -2b genes of strains and by determining 50% inhibitory concentrations of the seven agents for PBP1a, -2x, and -2b. Two PSSP strains had alterations in PBP2x (L5463V) (one strain) or PBP2b (T4453A) (one strain). All eight PISP strains had at least two alterations-T3383P or A or H3943Y in PBP2X and T4453A in BPB2b. All PRSP strains had the same changes seen in PISP strains, as well as T3713A or S substitutions in PBP1a. The two most resistant PRSP strains had a second change in PBP2x (M3393F) in a conserved motif. The afnities of penicillin and ampicillin for all three PBPs were decreased for PRSP and most PISP strains. The afnity of amoxicillin for PBP1a and -2x was decreased only for PRSP. Cefaclor and cefprozil showed decreased afnity of PRSP but not PISP for all three PBPs. Cefuroxime showed decreased afnity of PISP and PRSP for PBP1a and -2x but no change for PBP2b. Cefditoren showed no difference in PBP afnity based on penicillin or cefditoren MICs, indicating a different PBP target for this agent. Overall, the MICs for and PBP afnities of the strains correlated with the changes found in the PBP active binding sites. The worldwide incidence of infections caused by Streptococcus pneumoniae isolates resistant to penicillin and other antimicrobial agents has increased at an alarming rate during the past 2 decades (2). In a recent U.S. study, penicillin-nonsusceptible pneumococci were found in 50.4% of 1,476 strains, and the prevalence of macrolide-resistant strains was 65.6% in penicillin-resistant strains (20). The higher the penicillin MIC, the more likely it is that the strain will be multidrug resistant. Multidrug-resistant (including resistance to uoroquinolones) pneumococci have been reported in Hong Kong (19), Canada (7), and Spain (24), and the clonal spread of these strains from country to country and continent to continent is of concern. There is an urgent need for oral -lactams that can be used for outpatient treatment of infections, such as pneumonia, bronchitis, sinusitis, and otitis media, caused by penicillin- and macrolide-nonsusceptible pneumococci. Amoxicillin has excellent in vitro antipneumococcal activity (1, 3, 18) and has been shown to select for resistant laboratory mutants less frequently than other compounds (27, 29). Previous studies have documented the potent in vitro antipneumococcal activity of cefditoren, with MICs at which 50 and 90% of isolates are inhibited of 0.5 and 1.0 g/ml, respectively, against penicillin-resistant strains (35, 36). Cefditoren also selects for resistant laboratory
* Corresponding author. Mailing address: 500 University Dr., Hershey, PA 17033. Phone: (717) 531-5113. Fax: (717) 531-7953. E-mail: pappelbaum@psu.edu. 1273

mutants very rarely, comparably to amoxicillin (C. L. Clark, K. Nagai, B. E. Dewasse, G. A. Pankuch, L. M. Ednie, M. R. Jacobs, and P. C. Appelbaum, unpublished data). Penicillin-binding proteins (PBPs) are cell wall transpeptidases that catalyze the assembly of cell wall peptidoglycan by transpeptidation of the peptide side chains of murein units. Six PBPs are found in S. pneumoniae: the ve high-molecular-mass PBPs (PBP1a [79.7 kDa], PBP1b [89.6 kDa], PBP2x [82.3 kDa], PBP2a [80.8 kDa], and PBP2b [82.3 kDa]) and one low-molecular-mass PBP (PBP3 [45.2 kDa]) (1012, 1318). The high-molecular-mass PBPs are made up of an N-terminal hydrophobic region, a central penicillin-binding domain, and a C-terminal domain. The active site of transpeptidase activity is formed by three conserved amino acid motifs, SXXK, SXN, and KT(S)G. These motifs occur at amino acid positions 370 to 373, 428 to 430, and 557 to 559 in PBP1a, at positions 337 to 340, 395 to 397, and 547 to 549 in PBP2x, and at positions 385 to 388, 442 to 444, and 614 to 616 in PBP2b (17, 18). Changes in these motifs, or in the positions anking these motifs, are associated with low-afnity variants of the PBPs. These changes are the results of point mutations in strains or recombination of PBP genes with PBP genes of other pneumococci or of viridans streptococci to form mosaic genes. Penicillin resistance in S. pneumoniae is mediated by stepwise alterations of PBPs (18). Specically, decreased afnity of PBP1a, -2x, and -2b for -lactams has been reported to play an important part in resistance (46, 18, 23, 32). Alterations in the conserved

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ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Origins of 18 pneumococcal isolates tested in this study
Strain Sample no. Age of patient (yr) Site of isolation Country

motifs in PBP2b are associated with resistance to penicillin (14), and alterations in PBP2x mediate low-level resistance to cephalosporins (4, 8, 12, 14). Additional alterations in PBP1a raise penicillin G MICs to 1 g/ml and cefotaxime MICs to 0.5 g/ml (4, 26, 33, 37). These three PBPs are considered to be the key targets for these agents and were therefore chosen for examination in this study. The purpose of this study was to determine the association between PBP afnity for various -lactams and the gene sequences of the conserved motifs of selected PBPs, using a selection of penicillin-susceptible, -intermediate, and -resistant pneumococci. The -lactams used, penicillin G, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor, were chosen to represent commonly used therapeutic agents or, in the case of cefditoren, a new potent oral cephalosporin.
MATERIALS AND METHODS Strains. Eighteen recent clinical pneumococcal strains (four penicillin-susceptible [PSSP] strains for which the penicillin MICs were 0.06 g/ml, eight penicillin-intermediate [PISP] strains for which the penicillin MICs were 0.125 to 1.0 g/ml, and six penicillin-resistant [PRSP] strains for which the penicillin MICs were 2.0 g/ml) in our collection were used for this study. The strains were not clonally related and were all isolated from different geographic locations. NCCLS breakpoints were used for interpretation of penicillin G susceptibility categories (28). MIC testing. MIC testing was done by the agar dilution method using MuellerHinton agar with 5% debrinated sheep blood as previously described (35, 36). Plates were incubated for 20 h in air after inoculation. Susceptibility testing was performed for penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor. Amoxicillin was obtained from SmithKline Beecham (King of Prussia, Pa.), cefditoren was obtained from Meiji Seika Kaisha (Yokohama, Japan), and the other compounds were obtained from their respective manufacturers. Sequencing of pbp1a, -2x, and -2b genes by PCR. Template DNA for PCR was prepared using the Prep-A-Gene kit (Bio-Rad, Hercules, Calif.) as recommended by the manufacturer. A 1.2-kb segment of pbp1a was amplied using the primers and cycling conditions described by Asahi and Ubukata (5). A 2-kb segment of pbp2x and a 1.4-kb segment of pbp2b were amplied using primers and cycling conditions described previously (23, 38). PCR products were puried from primers and excess nucleotides using a QIAquick PCR purication kit (Qiagen, Valencia, Calif.) as recommended by the manufacturer and were sequenced directly with an Applied Biosystems model 373A DNA sequencer using the following additional primers: for pbp1a, 5 -2377GCAAGTAGTGAAAAGA TGGCT2400-3 , 5 -2545TGTCGGTCATCATATAGGC2527-3 , and 5 -2963GATT GTGAAGTTGAACTATCTG2944-3 ; for pbp2x, 5 -991GGAACAGAACAAGT TTCCCAAC1112-3 , 5 -1354GATGCCACGATTCGAGATTGGG1375-3 , 5 1645TTTACAGCTATTGCTATTGATGG1667-3 , 5 -1488CCAGGTAGCATCT CCCAT1471-3 , and 5 -2105AGAGAGTCTTTCATAGCTGAAGC2083-3 ; for pbp2b, 5 -1196TTGCTGAAAAGTTATTTCAATTC1218-3 , 5 -1466ATTGTCTT CCAAGGTTCAGCT1486-3 , 5 -1775TTCCTTTGGGCAGTTTGATAAC17943 , and 5 -CAACAATACGAGGAGCCACA-3 . The results of DNA sequencing were aligned using Vector NTI 6.0 (Infomax, Inc., Bethesda, Md.) and compared to DNA and amino acid sequences from the penicillin-susceptible S. pneumoniae R6 strain as previously reported (10, 15, 21, 22, 25, 31, 37, 39). PBP binding assays. PBP binding assays were performed as described previously (27). Five hundred microliters of early-log-phase culture was centrifuged for 3 min at 5,000 g. The cells were resuspended in 0.1 M phosphate buffer containing unlabeled drug and incubated for 15 min at 37C. [3H]benzylpenicillin (2 Ci; Amersham Life Sciences, Piscataway, N.J.) was added, and the mixture was incubated for 15 min at 37C. the cells were lysed by the addition of 0.1 M phosphate buffer with 0.1% Triton X-100. A 10-fold excess of unlabeled benzylpenicillin was added, and the mixture was incubated for 15 min at 37C. Samples were resuspended in 40 l of lysis buffer (2% sodium dodecyl sulfate, 4% 2-mercaptoethanal, 10% glycerol, and 0.002% bromphenol blue in 1 M Tris [pH 6.8] buffer) and heated to 100C for 3 min. Labeled PBPs were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 10% gel and visualized by uorography using preashed Hyperlm MP (Amersham Pharmacia Biotech, Piscataway, N.J.). PBP bands were interpreted by determining 50%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

49 768 875 891 731 737 914 714 420 850 755 866 997 703 500 799 810 775

Unknown 2 3 78 1 4 1 12 Unknown 41 87 1 Unknown 3 Unknown 3 56 22

Unknown Maxillary sinus Eye Sputum Ear Maxillary sinus Ear Blood Nasopharynx Sputum Eye Eye Blood Ear Blood Nasopharynx Sputum Nasopharynx

United States Mexico United United United United States States States States

United States United States Bulgaria United States United States United States Canada Mexico Korea United States United States United States

inhibitory concentrations (IC50s) for PBP1a, PBP2x, and PBP2b bands as follows. Fluorograms were converted to electronic format using a video camera, and the IC50s of each agent were determined for the PBP bands based on the intensity of each band using Molecular Analyst software (Bio-Rad, Inc.). IC50s were dened as the concentration of each agent that resulted in a 50% decrease in intensity of PBP bands on uorograms compared to controls without the addition of unlabeled competitive compounds (30, 31). Saturation of PBPs by radiolabeled benzylpenicillin in competition experiments was addressed as described previously (30, 31). Statistical analysis. Geometric mean MICs and correlation coefcients were determined using the statistical functions of Microsoft Excel 2000 (Microsoft Corporation, Redmond, Wash.).

RESULTS Susceptibility of pneumococcal strains. The origins of 18 clinical pneumococcal isolates are shown in Table 1. Clinical isolates from various countries and patients were chosen for this study. The MICs of the pneumococcal strains tested are shown in Table 2. The geometric mean values of MICs (and MIC ranges) in micrograms per milliliter for four PSSP strains were as follows: penicillin, 0.04 (0.03 to 0.06); amoxicillin, 0.03 (0.015 to 0.03); ampicillin, 0.08 (0.06 to 0.25); cefditoren, 0.03 (0.015 to 0.06); cefuroxime, 0.15 (0.06 to 0.25); cefprozil, 0.15 (0.06 to 0.5); and cefaclor, 0.6 (0.25 to 1). Those for eight PISP strains were as follows: penicillin, 0.25 (0.125 to 1); amoxicillin, 0.2 (0.06 to 1); ampicillin, 0.25 (0.06 to 1); cefditoren, 0.1 (0.03 to 0.25); cefuroxime, 1 (0.25 to 4); cefprozil, 0.8 (0.25 to 2); and cefaclor, 1.4 (0.5 to 8). Those for six PRSP strains were as follows: penicillin, 2.8 (2 to 4); amoxicillin, 2.5 (1 to 8); ampicillin, 6.3 (2 to 16); cefditoren, 1.3 (0.5 to 4); cefuroxime, 10.1 (4 to 32); cefprozil, 20.2 (16 to 32); cefaclor, 160 (64 to 256). The mean MICs for PISP were generally 2- to 6-fold higher than those for PSSP, whereas the geometric mean MICs for PRSP were generally 40- to 80-fold higher than those for PSSP and 10- to 25-fold higher than for PISP. However, there were some exceptions to these ndings, with the mean MICs of cefprozil and cefaclor for PRSP being over 100-fold higher than the mean MICs for PSSP and those of cefaclor for PRSP being over 100-fold higher than the mean MICs for PISP.

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TABLE 2. MICs of seven agents against 18 study strains

Strain MIC ( g/ml) Penicillin G Amoxicillin Ampicillin Cefditoren Cefuroxime Cefprozil Cefaclor

PSSP 1 2 3 4 PISP 5 6 7 8 9 10 11 12 PRSP 13 14 15 16 17 18

0.03 0.06 0.06 0.03 0.125 0.125 0.5 1 0.25 0.125 0.25 0.25 2 4 2 2 4 4

0.03 0.015 0.03 0.03 0.06 0.125 0.125 0.125 0.25 0.125 1 0.5 1 1 2 2 8 8

0.06 0.06 0.25 0.06 0.06 0.125 0.125 0.25 0.5 0.125 1 1 8 2 8 2 16 16

0.015 0.015 0.06 0.06 0.03 0.03 0.25 0.25 0.06 0.06 0.25 0.125 1 1 1 0.5 2 4

0.06 0.125 0.25 0.25 0.5 0.25 1 4 0.5 2 2 1 8 8 8 4 6 32

0.125 0.06 0.5 0.125 0.5 0.25 0.5 1 1 2 2 1 16 16 16 16 32 32

1 0.25 1 0.5 0.5 0.5 0.5 4 1 8 2 2 128 64 128 64 256 256

DNA sequencing and amino acid alterations in PBP1a, -2x, and -2b. The amino acid sequences of the three conserved PBP motifs of the three PBPs studied are shown in Table 3. There were no changes in the conserved motifs of PBP1a for PSSP or PISP, while all PRSP had T3713A or S substitutions in the STMK motif and no changes in the other two PBP1a motifs. The STMK motif of PBP2x showed no changes in PSSP

strains, while T3383A or P substitutions were found in six of eight PISP and in all PRSP strains. Additionally, the two most resistant PRSP strains (strains 17 and 18) showed M3393F substitutions. Changes in the amino acid before the other two motifs of PBP2x were found in two PISP strains for the SSN motif (H3943Y) and for one PSSP, two PISP, and all PRSP strains for the KSG motif (L5463V).

TABLE 3. Sequences of the three conserved amino acid motifs of PBPs 1a, 2x, and 2b that form the active penicillin-binding site cavities of the PBPs in S. pneumoniae R6 and the 18 study strains (sequences anking these motifs are also shown where relevant)
Changes in amino acids of conserved PBP sites making up active penicillin-binding site of PBP: Strain 370373a 1a 428430 557559 337340 2x 394397 546549 385388 2b 442445 614616

R6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
a b c

STMK c SAMKd SAMK SAMK SAMK SSMK SSMK

SRN

KTG

STMK SPMK SPMK SAMK SAMK SAMK SAMK SAMK SAMK SAMK SAMK SAFK SAFK

HSSN YSSN YSSN

LKSG VKSG VKSG VKSG VKSG VKSG VKSG VKSG VKSG VKSG

SVVK

SSNT SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA SSNA

KTG

Amino acid position numbers of amino acids shown in column below. For S. pneumoniae R6, conserved amino acid motifs are shown in boldface and anking amino acids (where applicable) are shown in regular typeface. , no change in amino acid motifs of test strains from those of S. pneumoniae R6. d Changes in amino acid motifs of test strains from those of S. pneumoniae R6 are shown in boldface for study strains.

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FIG. 1. Correlation between MICs and IC50s for PBPs 1a, 2x, and 2b of PSSP (}), PISP (F), and PRSP () for the agents tested.

The only change found in the motifs of PBP2b were a T4443A substitution in the position following the central SSN motif. This change was found in one PSSP and all PISP and PRSP strains. Other amino acid changes noted were a Y insertion in PBP2b between positions 429 and 430 in PISP strain 10. There were many other amino acid changes at other positions not known to be associated with the active binding sites of these enzymes. Afnities of agents tested for PBP1a, -2x, and -2b. Graphs of MICs versus IC50s for PBP1a, -2x, and -2b of the antimicrobial agents for the 18 pneumococcal strains tested are shown in Fig. 1. Overall, the afnity of penicillin for each PBP generally

decreased as the MICs increased, but overall agreement was poor (R2 0.23 for PBP1a, 0.27 for PBP2x, and 0.12 for PBP2b). Ampicillin showed a more striking correlation between PBP afnity and MICs for all three PBPs, with a more linear relationship for all three PBPs tested than was found with the other agents tested in this study (R2 0.76 for PBP1a, 0.65 for PBP2x, and 0.62 for PBP2b). Amoxicillin showed a different afnity pattern, with a bimodal pattern for all three PBPs separating most of the strains for which the amoxicillin MICs were 1 g/ml from those for which the amoxicillin MICs were 1 g/ml. The amoxicillin MICs of PRSP strains 14 and 15 (both 1 g/ml) did not appear to be related to afnity for PBP2x or PBP2b (IC50s, 0.1 to 0.15 g/ml). Cefaclor

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FIG. 1Continued.

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showed a clear bimodal distribution separating PSSP-PISP strains from PRSP strains. Cefprozil showed a bimodal distribution of PBP afnities similar to that of cefaclor, but the decrease in PBP afnities was disproportionate to the rise in MICs, suggesting another target for this agent. Cefuroxime showed a pattern different from that seen with the other agents studied, with linear correlation between afnities and MICs for PBP1a (R2 0.54) and -2x (R2 0.52) but no relationship for PBP2b. In fact, the afnities of PISP and PRSP strains for PBP2b were higher than those of some PSSP strains. Cefditoren showed no clear association between PBP afnities and MICs, and increases in MICs did not appear related to afnities for the three PBPs tested. Correlation of MICs, PBP mutations, and PBP afnities. The associations shown in this study between MIC changes and PBP afnity changes show which agents bind to the PBPs studied and whether the PBP afnities of these agents change as the MICs rise. However, correlation of these changes with changes in the conserved motifs of the PBP binding sites was more complex. Nevertheless, it appears that the single changes detected either in the STMK motif or anking the SSN motifs of PBP2x are associated with decreased susceptibility and afnity of PISP strains for penicillin, and addition of a change in the STMK motif of PBP1a is associated with decreased susceptibility and afnity of PRSP strains. No changes in any of the three motifs in PBP1a were detected in PSSP or PISP strains. The changes found in the amino acids preceding the SSN motif of PBP2x (H3943Y) and following the SSN motif of PBP2b (T4453A) show no clear associations with the penicillin MIC or afnity changes but could contribute to resistance when present with changes in other key motifs of the PBPs. DISCUSSION PBP1a, -2x, and -2b are generally recognized as the major PBPs associated with the activities of penicillins and some cephalosporins (e.g., cefaclor and cefprozil), and PBP1a and -2x are generally recognized as the major PBPs associated with those of other cephalosporins (e.g., cefotaxime and cefuroxime) (15, 17). However, other PBPs and other mechanisms, such as altered stem peptide (mur) genes, altered histidine protein kinase CiaH, and altered glycosyltransferase CpoA, can also affect -lactam activity (16, 21, 34). The low-afnity variants of PBPs are the results of recombination of the genes coding for these proteins with genes of other species, such as viridans streptococci. Much work has been done on laboratory mutants with dened point mutations in PBPs, but these relationships are more difcult to determine in clinical isolates. Laboratory transformation experiments have shown that lowafnity forms of PBP2b and -2x confer low-level penicillin resistance and are prerequisites for high-level resistance (12, 14, 17, 22). High-level penicillin resistance requires the presence of a low-afnity form of PBP1a as well as either lowafnity PBP2x or low-afnity PBP2x and -2b (17, 33). Our ndings were in general agreement with these reports, with the afnities of isolated PBPs, as well as the PBP active binding site genes, conrming these relationships. The changes we found in the STMK motif of PBP2x (T3383A or P substitution) are identical to those previously reported (4, 8). However, the two most resistant strains we

studied, strains 17 and 18, had an additional M3393F substitution, which has been reported in clinical isolates of cefotaxime-resistant strains in Japan (cefotaxime MICs, 2 to 8 g/ml) and the United States (cefotaxime MICs, 8 to 32 g/ml) (4, 8). These two strains were also remarkable for having higher amoxicillin and ampicillin MICs than penicillin MICs and very high cefaclor MICs ( 256 g/ml). These two amino acid substitutions in the STMK motif of PBP2x have been shown to increase cefotaxime MICs from 0.016 to 1 g/ml in pneumococcal transformants (4). Additional strains need to be examined to determine the signicance of this nding. Hakenbeck and coworkers also reported PBP2x T5503A and Q5523E substitutions, which are close to the K547SG motif, to be associated with cephalosporin resistance in a laboratory mutant of pneumococci (17). We found no T550 substitutions and could not conrm this association. However, we did nd Q5523E substitutions in two PSSP and three PISP without signicant cephalosporin resistance in these strains, suggesting that the reported association may not be valid in clinical isolates. The PBP2b T4453A substitution found in all PISP and PRSP and one PSSP has been found in all low-level -lactamresistant pneumococci examined, as well as in Streptococcus mutans (11, 14). A PBP1a with a T3713A or S substitution has also been associated with decreased -lactam afnity of this PBP and high-level penicillin resistance, as we found in this study (4, 15, 33). A 4-amino-acid substitution in PBP1a, T574SQF3NTGY, has also been described as being important in PBP1a with respect to the interaction with penicillin and the development of resistance (33). We found this change in strains 7, 9, 10, and 12 to 18 in this study, but we are unsure of its signicance, as this change was not near one of the PBP penicillin-binding sites and showed no clear correlation with susceptibility results. Our study has extended our knowledge of the complex interrelationships among MICs, PBP afnities, and PBP binding site changes, utilizing clinical isolates. Single-amino-acid substitutions in or anking the STMK motif of PBP2x and the SSN motif of PBP2b appear to be the predominant changes in PISP strains. A substitution in the STMK motif of PBP1a, in addition to the changes associated with PISP strains, appears to correlate with PRSP strains. An intriguing nding of our study is a second substitution in the STMK motif of PBP2x, associated with the most resistant strains and strains with higher aminopenicillin than penicillin MICs. The associations demonstrated among MICs, PBP afnities, and PBP structures are useful in illustrating the mechanisms underlying the MIC variations of the -lactams studied. In particular, these associations may explain the improved activity of amoxicillin compared to penicillin against many PISP strains and the higher MICs of cefuroxime, cefprozil, and cefaclor against PRSP strains. However, we have no explanation for the lack of correlation between PBP afnities and MICs for cefditoren. The afnity of amoxicillin for PBP2b decreased for PISP and PRSP strains, but the afnity of this agent for PBP1a and -2x did not decrease for most PISP strains, in agreement with the amoxicillin MICs (0.125 g/ml) for some PISP, such as strains 7 and 8, being lower than the penicillin MICs (0.5 to 1 g/ml). Substitutions outside the specic areas examined might also contribute to resistance, either by providing compensatory

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structural alterations stabilizing the primary active-site mutation or by alterations further affecting the active-site structure and function directly via a distance effect. In addition, other mechanisms are involved in the activities of -lactams in pneumococci. Filipe and Tomasz (13) have postulated that more distant mutations, such as murM and murN genes, which code for stem peptides that cross-link peptidoglycan chains, might play a role in the expression of penicillin resistance in pneumococci. These aspects were not explored in the present study. Additionally, it is recognized that the methodological limitations inherent in IC50 determinations might have limited our ability to correlate these results with MICs. Also, what is actually measured in competition assays is complex and includes acylation, deacylation, and binding reactions. Additional experiments, such as transformation of modied PBPs from selected strains with raised penicillin MICs into susceptible strains to see if the PBP changes are responsible for the level of resistance found in clinical isolates may also yield valuable information. We should not forget that these relationships are not the only determinants of the clinical relevance of these agents, which is also determined by the pharmacokinetic parameters achieved with the dosing regimen chosen and the pharmacodynamic properties of the agent (9).
ACKNOWLEDGMENTS This study was supported in part by grants from Glaxo SmithKline Pharmaceuticals (King of Prussia, Pa.) and TAP Pharmaceutical Products, Inc. (Lake Forest, Ill.). We thank Kimiko Ubukata for her critical comments on the manuscript.
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