This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kosowska-Shick, K.
Right arrow Articles by Appelbaum, P. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kosowska-Shick, K.
Right arrow Articles by Appelbaum, P. C.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, May 2009, p. 2176-2180, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01566-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Binding of Faropenem and Other β-Lactam Agents to Penicillin-Binding Proteins of Pneumococci with Various β-Lactam Susceptibilities{triangledown}

Klaudia Kosowska-Shick, Pamela McGhee, and Peter C. Appelbaum*

Department of Pathology, Hershey Medical Center, Hershey, Pennsylvania

Received 24 November 2008/ Returned for modification 6 February 2009/ Accepted 11 February 2009


arrow
ABSTRACT
 
Faropenem demonstrated low MICs (≤1 µg/ml) for all penicillin-susceptible and nonsusceptible pneumococci and exhibited very strong abilities to bind to Streptococcus pneumoniae penicillin-binding proteins (PBPs), except for PBP2X. The lower faropenem affinity for PBP2X did not affect MICs for any strains tested, and only imipenem had lower MICs, with much lower binding affinities for all PBPs tested, than faropenem.


arrow
INTRODUCTION
 
The mechanism of increased β-lactam MICs for Streptococcus pneumoniae strains is sequential alterations in the penicillin-binding proteins (PBPs). Six PBPs are found in S. pneumoniae: five high-molecular-mass PBPs (PBP1A, PBP1B, PBP2X, PBP2A, and PBP2B) and one low-molecular-mass PBP, PBP3. It is well known that these different PBPs have different affinities for β-lactams (1, 7, 9, 11, 21, 26). The most highly penicillin G-resistant pneumococcal clinical isolates (for which MICs are 2 to 16 µg/ml) produce altered forms of PBP1A, PBP2X, PBP2B, and PBP2A that have reduced drug affinities (13, 18, 20, 27). Altered PBPs in pneumococci have greatly decreased affinities for almost all β-lactams, including expanded-spectrum cephalosporins (27).

The active sites of PBPs are formed by three conserved amino acid motifs, SXXK, SXN, and KT(S)G. Changes in these motifs or in the positions flanking these motifs are associated with low-affinity PBP variants (21). Alterations in the conserved motifs in PBP2B are associated with resistance to penicillin G, and alterations in PBP2X mediate low-level resistance to cephalosporins. Additional alterations in PBP1A raise penicillin G MICs to ≥1 µg/ml and cefotaxime MICs to ≥0.5 µg/ml (21). Davies et al. have also observed that carbapenems have high affinities for all PBPs in a penicillin-susceptible isolate but show reduced binding affinities for PBPs, particularly PBP2X and PBP2B, in a penicillin-resistant isolate (9).

Among all PBPs, pneumococcal PBP2X has one of the highest affinities for penicillin G (19). Zhao et al. have also suggested that PBP2A is a naturally resistant form of PBP and that PBP2A may be able to take over the activities of other PBPs in the presence of clinically relevant concentrations of β-lactam antibiotics, which may therefore account for its role in resistance to β-lactams (18, 30). Additionally, the presence of a low-affinity PBP1A is essential for high-level resistance but requires a modified PBP2B and/or PBP2X (20, 23). Since the expanded-spectrum cephalosporins do not interact with PBP2B (15), only PBP2X and PBP1A play a role in resistance to these compounds (20).

Faropenem is an oral penem with excellent activity against the primary bacterial pathogens responsible for community-acquired respiratory tract infections, including non-penicillin-susceptible S. pneumoniae, β-lactamase-producing Haemophilus influenzae, and β-lactamase-producing Moraxella catarrhalis isolates (6, 12, 25, 28). Faropenem demonstrates lower MICs (MIC90, 1 µg/ml) than other β-lactams and macrolides for panresistant non-vaccine serotype 19A S. pneumoniae strains, which are spreading in the United States (5, 22).

In the present study, we examined the antipneumococcal activities of faropenem by comparing the affinities of faropenem for all pneumococcal PBPs in strains with different β-lactam resistance phenotypes with those of representatives of the penicillin (penicillin G and amoxicillin), carbapenem (imipenem), and cephem (cefuroxime) classes.

Twelve clinical, clonally unrelated pneumococcal isolates from different countries (Table 1) classified by penicillin G susceptibility according to old CLSI criteria (4) (three penicillin G susceptible, three penicillin G intermediate, and six penicillin G resistant) were tested by the CLSI macrodilution method (3, 4). Fragments of the genes pbp1A (nucleotide region 870 to 1950), encoding 350 amino acids; pbp2B (nucleotide region 655 to 2028), encoding 458 amino acids; and pbp2X (nucleotide region 301 to 2034), encoding 578 amino acids were amplified using primers and conditions described previously (17, 24) and directly sequenced with a CEQ8000 genetic analysis system (Beckman Coulter, Fullerton, CA). The obtained sequences were analyzed by BLAST and ClustalW (29). PBP alleles were identified based on comparison to sequences of analyzed S. pneumoniae R6 genes and named with an alphabet letter based on homology to the R6 sequence, e.g., allele A has the highest degree of homology to the R6 sequence, and allele L has the lowest degree of homology to the R6 sequence. It was noticed that numbering for the PBP2B amino acid sequence was off by 6 amino acids compared to that of the original S. pneumoniae R6 sequence (accession number AE008520.1) (16). Previous reports in the literature have described PBP2B motifs which were off by the above-mentioned 6 amino acids, and because of this finding, we have retained the old amino acid numbering to be able to correlate mutations (10, 17, 21).


View this table:
[in this window]
[in a new window]

  		TABLE 1. MICs determined for all strains tested in our studya

Whole cells of S. pneumoniae were used for PBP labeling with Bocillin FL (Invitrogen, Carlsbad, CA), and PBPs were visualized using an imager (Bio-Rad, Hercules, CA). The 50% inhibitory concentration (IC50) values were determined using Quantity One software (Bio-Rad) (26).

MICs (in micrograms per milliliter) for all strains, sequencing analyses, and IC50s for all PBPs from all strains tested can be seen in Tables 1, 2, and 3.


View this table:
[in this window]
[in a new window]

  		TABLE 2. Amino acid alterations in the conserved motifs of PBP1A, PBP2X, and PBP2B in 13 tested strains compared to S. pneumoniae R6 PBP sequences


View this table:
[in this window]
[in a new window]

  		TABLE 3. IC50s of antibiotics and allele identification for all strains tested

All PBPs, except for PBP3 proteins, from three penicillin G-resistant strains showed no demonstrable binding affinities (Table 1). PBP2A proteins from two strains, one with intermediate resistance to penicillin G and one penicillin G susceptible, showed no binding affinities (Table 3). No specific mutations were present in the pbp genes of the latter strains, and we are unable to explain this phenomenon without additional tests.

Faropenem exhibited the lowest MICs for three pneumococci in which all PBPs, except PBP3, showed no detectable affinities (Table 1). Imipenem had lower MICs than faropenem for all nine strains in which PBPs other than PBP3 also had demonstrable binding affinities. Penicillin G, amoxicillin, and cefuroxime demonstrated much higher MICs than those of faropenem and imipenem, especially for penicillin G-intermediate and -resistant strains. Sequencing analyses of pbp1A, pbp2B, pbp2X, and pbp3 showed that PBP1A had the most altered amino acid sequences compared to the S. pneumoniae R6 sequence (with 85 to 99% similarity), followed by PBP2X (86 to 100% similarity), PBP2B (89 to 96% similarity), and PBP3 (99% similarity). Biçmen et al. (2) and del Campo et al. (10) have described a correlation which differs from our findings in that their most variable alleles were those for PBP2X, followed by those for PBP2B and PBP1A. Amino acid substitutions found in and adjacent to the PBP1A, PBP2X, and PBP2B motifs SXXK, SXN, and KT/SG have been described previously (1, 2, 8, 9, 11, 14, 17, 21, 24) (Table 2).

The accumulation of alterations in PBP2B compared to the R6 amino acid sequence correlated with increasing faropenem MICs. In contrast, amino acid alterations in other PBPs did not show such correlation. Increases of cefuroxime, amoxicillin, and penicillin MICs correlated with higher percentages of alterations in all PBPs tested; such correlations have been observed before (9, 14, 24). Changes in imipenem MICs did not correlate with alterations in all PBPs or binding affinities for PBPs, and among all the antibiotics in the study, imipenem had the lowest affinities for all PBPs tested except for PBP2B. Faropenem demonstrated the highest binding affinities for PBP1B, PBP2B, and PBP3 of all the PBPs and the lowest affinity for PBP2X. Dalhoff et al. have observed a similar correlation for faropenem-binding affinity, which was highest for PBP1, followed by PBP2 and PBP3 (7). Lower faropenem affinity for PBP2X did not affect MICs for any strains tested. In contrast, even though we observed a high affinity of faropenem for PBP2B, amino acid alterations were still sufficient to increase faropenem MICs but to no higher than 1 µg/ml. As expected, cefuroxime had the highest affinities for PBP1A, PBP2A, and PBP2X of all the PBPs, because cephalosporins do not use PBP2B as a primary target (9, 14). The decreased binding affinities of amoxicillin and penicillin G for PBP2B reflect an increase in the MICs of the drugs, which agrees with the fact that PBP2B is thought to be the primary target for carbapenems and penicillins (24). Similar correlations have been described previously (9, 21).

Amoxicillin, penicillin, and cefuroxime MIC increases were much more affected by the accumulation of alterations in PBPs than those of imipenem and faropenem. Interestingly, imipenem had the lowest MICs for all strains in which PBPs other than PBP3 had detectable binding affinities, despite imipenem's having the lowest binding affinities for all PBPs (except for PBP2B) of the antibiotics tested. Faropenem had the lowest MICs for three strains in which all PBPs except PBP3 showed no demonstrable binding affinities, which may indicate a promising property for the treatment of multiple-drug-resistant S. pneumoniae isolates.

In conclusion, we have shown that faropenem efficiently inhibited essential S. pneumoniae PBPs with high affinities for all PBPs except PBP2X. This compound represents a potential therapeutic alternative for pediatric infections such as otitis media caused by panresistant pneumococcal type 19A, which are resistant to all drugs currently approved by the FDA for pediatrics (22) and are being treated (for want of alternatives) with respiratory quinolones such as levofloxacin and moxifloxacin or linezolid.


arrow
Nucleotide sequence accession numbers.
 
All sequences obtained for pbp1a, pbp2X, and pbp2B were deposited in GenBank. Accession numbers are listed in Table 2.


arrow
ACKNOWLEDGMENTS
 
This study was supported by a grant from Replidyne, Inc., Louisville, CO.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: 500 University Dr., Hershey, PA 17033. Phone: (717) 531-5113. Fax: (717) 531-7953. E-mail: pappelbaum{at}psu.edu Back

{triangledown} Published ahead of print on 23 February 2009. Back


arrow
REFERENCES
 
    1
  1. Asahi, Y., and K. Ubukata. 1998. Association of a Thr-371 substitution in a conserved amino acid motif of penicillin-binding protein 1A with penicillin resistance of Streptococcus pneumoniae. Antimicrob. Agents Chemother. 42:2267-2273.[Abstract/Free Full Text]
    2. 2
  2. Biçmen, M., Z. Gulay, S. V. Ramaswamy, D. M. Musher, and D. Gur. 2006. Analysis of mutations in the pbp genes of penicillin-non-susceptible pneumococci from Turkey. Clin. Microbiol. Infect. 12:150-155.[CrossRef][Medline]
    3. 3
  3. CLSI. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 7th ed. Approved standard M7-A7. CLSI, Wayne, PA.
    4. 4
  4. CLSI. 2006. Performance standards for antimicrobial susceptibility testing: 17th informational supplement M100-S17. CLSI, Wayne, PA.
    5. 5
  5. Critchley, I. A., M. R. Jacobs, S. D. Brown, M. M. Traczewski, G. S. Tillotson, and N. Janjic. 2008. Prevalence of serotype 19A Streptococcus pneumoniae among isolates from U.S. children in 2005-2006 and activity of faropenem. Antimicrob. Agents Chemother. 52:2639-2643.[Abstract/Free Full Text]
    6. 6
  6. Dalhoff, A., N. Janjic, and R. Echols. 2006. Redefining penems. Biochem. Pharmacol. 71:1085-1095.[CrossRef][Medline]
    7. 7
  7. Dalhoff, A., T. Nasu, and K. Okamoto. 2003. Target affinities of faropenem to and its impact on the morphology of gram-positive and gram-negative bacteria. Chemotherapy 49:172-183.[CrossRef][Medline]
    8. 8
  8. Davies, T. A., M. G. Page, W. Shang, T. Andrew, M. Kania, and K. Bush. 2007. Binding of ceftobiprole and comparators to the penicillin-binding proteins of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Antimicrob. Agents Chemother. 51:2621-2624.[Abstract/Free Full Text]
    9. 9
  9. Davies, T. A., W. Shang, K. Bush, and R. K. Flamm. 2008. Affinity of doripenem and comparators to penicillin-binding proteins in Escherichia coli and Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 52:1510-1512.[Abstract/Free Full Text]
    10. 10
  10. del Campo, R., F. Cafini, M. I. Morosini, A. Fenoll, J. Linares, L. Alou, D. Sevillano, R. Canton, J. Prieto, and F. Baquero. 2006. Combinations of PBPs and MurM protein variants in early and contemporary high-level penicillin-resistant Streptococcus pneumoniae isolates in Spain. J. Antimicrob. Chemother. 57:983-986.[Abstract/Free Full Text]
    11. 11
  11. Grebe, T., and R. Hakenbeck. 1996. Penicillin-binding proteins 2b and 2x of Streptococcus pneumoniae are primary resistance determinants for different classes of beta-lactam antibiotics. Antimicrob. Agents Chemother. 40:829-834.[Abstract]
    12. 12
  12. Hadley, J. A., G. S. Tillotson, R. Tosiello, and R. M. Echols. 2006. Faropenem medoxomil: a treatment option in acute bacterial rhinosinusitis. Expert Rev. Anti-Infect. Ther. 4:923-937.[CrossRef]
    13. 13
  13. Hakenbeck, R., H. Ellerbrok, T. Briese, S. Handwerger, and A. Tomasz. 1986. Penicillin-binding proteins of penicillin-susceptible and -resistant pneumococci: immunological relatedness of altered proteins and changes in peptides carrying the beta-lactam binding site. Antimicrob. Agents Chemother. 30:553-558.[Abstract/Free Full Text]
    14. 14
  14. Hakenbeck, R., A. Konig, I. Kern, M. van der Linden, W. Keck, D. Billot-Klein, R. Legrand, B. Schoot, and L. Gutmann. 1998. Acquisition of five high-Mr penicillin-binding protein variants during transfer of high-level β-lactam resistance from Streptococcus mitis to Streptococcus pneumoniae. J. Bacteriol. 180:1831-1840.[Abstract/Free Full Text]
    15. 15
  15. Hakenbeck, R., S. Tornette, and N. F. Adkinson. 1987. Interaction of non-lytic beta-lactams with penicillin-binding proteins in Streptococcus pneumoniae. J. Gen. Microbiol. 133:755-760.[Abstract/Free Full Text]
    16. 16
  16. Hoskins, J., W. E. Alborn, Jr., J. Arnold, L. C. Blaszczak, S. Burgett, B. S. DeHoff, S. T. Estrem, L. Fritz, D. J. Fu, W. Fuller, C. Geringer, R. Gilmour, J. S. Glass, H. Khoja, A. R. Kraft, R. E. Lagace, D. J. LeBlanc, L. N. Lee, E. J. Lefkowitz, J. Lu, P. Matsushima, S. M. McAhren, M. McHenney, K. McLeaster, C. W. Mundy, T. I. Nicas, F. H. Norris, M. O'Gara, R. B. Peery, G. T. Robertson, P. Rockey, P. M. Sun, M. E. Winkler, Y. Yang, M. Young-Bellido, G. Zhao, C. A. Zook, R. H. Baltz, S. R. Jaskunas, P. R. Rosteck, Jr., P. L. Skatrud, and J. I. Glass. 2001. Genome of the bacterium Streptococcus pneumoniae strain R6. J. Bacteriol. 183:5709-5717.[Abstract/Free Full Text]
    17. 17
  17. Kosowska, K., M. R. Jacobs, S. Bajaksouzian, L. Koeth, and P. C. Appelbaum. 2004. Alterations of penicillin-binding proteins 1A, 2X, and 2B in Streptococcus pneumoniae isolates for which amoxicillin MICs are higher than penicillin MICs. Antimicrob. Agents Chemother. 48:4020-4022.[Abstract/Free Full Text]
    18. 18
  18. Laible, G., B. G. Spratt, and R. Hakenbeck. 1991. Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Mol. Microbiol. 5:1993-2002.[Medline]
    19. 19
  19. Maurer, P., B. Koch, I. Zerfass, J. Krauss, M. van der Linden, J. M. Frere, C. Contreras-Martel, and R. Hakenbeck. 2008. Penicillin-binding protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodelling an essential enzyme into a resistance determinant. J. Mol. Biol. 376:1403-1416.[CrossRef][Medline]
    20. 20
  20. Munoz, R., C. G. Dowson, M. Daniels, T. J. Coffey, C. Martin, R. Hakenbeck, and B. G. Spratt. 1992. Genetics of resistance to third-generation cephalosporins in clinical isolates of Streptococcus pneumoniae. Mol. Microbiol. 6:2461-2465.[Medline]
    21. 21
  21. Nagai, K., T. A. Davies, M. R. Jacobs, and P. C. Appelbaum. 2002. Effects of amino acid alterations in penicillin-binding proteins (PBPs) 1a, 2b, and 2x on PBP affinities of penicillin, ampicillin, amoxicillin, cefditoren, cefuroxime, cefprozil, and cefaclor in 18 clinical isolates of penicillin-susceptible, -intermediate, and -resistant pneumococci. Antimicrob. Agents Chemother. 46:1273-1280.[Abstract/Free Full Text]
    22. 22
  22. Pichichero, M. E., and J. R. Casey. 2007. Emergence of a multiresistant serotype 19A pneumococcal strain not included in the 7-valent conjugate vaccine as an otopathogen in children. JAMA 298:1772-1778.[Abstract/Free Full Text]
    23. 23
  23. Reichmann, P., A. Konig, A. Marton, and R. Hakenbeck. 1996. Penicillin-binding proteins as resistance determinants in clinical isolates of Streptococcus pneumoniae. Microb. Drug Resist. 2:177-181.[Medline]
    24. 24
  24. Sanbongi, Y., T. Ida, M. Ishikawa, Y. Osaki, H. Kataoka, T. Suzuki, K. Kondo, F. Ohsawa, and M. Yonezawa. 2004. Complete sequences of six penicillin-binding protein genes from 40 Streptococcus pneumoniae clinical isolates collected in Japan. Antimicrob. Agents Chemother. 48:2244-2250.[Abstract/Free Full Text]
    25. 25
  25. Schurek, K. N., R. Wiebe, J. A. Karlowsky, E. Rubinstein, D. J. Hoban, and G. G. Zhanel. 2007. Faropenem: review of a new oral penem. Expert Rev. Anti-Infect. Ther. 5:185-198.[CrossRef]
    26. 26
  26. Sifaoui, F., M. D. Kitzis, and L. Gutmann. 1996. In vitro selection of one-step mutants of Streptococcus pneumoniae resistant to different oral β-lactam antibiotics is associated with alterations of PBP2x. Antimicrob. Agents Chemother. 40:152-156.[Abstract]
    27. 27
  27. Spratt, B. G. 1994. Resistance to antibiotics mediated by target alterations. Science 264:388-393.[Abstract/Free Full Text]
    28. 28
  28. Stone, K. C., R. Dagan, A. Arguedas, E. Leibovitz, E. Wang, R. M. Echols, N. Janjic, and I. A. Critchley. 2007. Activity of faropenem against middle ear fluid pathogens from children with acute otitis media in Costa Rica and Israel. Antimicrob. Agents Chemother. 51:2230-2235.[Abstract/Free Full Text]
    29. 29
  29. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]
    30. 30
  30. Zhao, G., T. I. Meier, J. Hoskins, and K. A. McAllister. 2000. Identification and characterization of the penicillin-binding protein 2a of Streptococcus pneumoniae and its possible role in resistance to β-lactam antibiotics. Antimicrob. Agents Chemother. 44:1745-1748.[Abstract/Free Full Text]

Antimicrobial Agents and Chemotherapy, May 2009, p. 2176-2180, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01566-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kosowska-Shick, K.
Right arrow Articles by Appelbaum, P. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kosowska-Shick, K.
Right arrow Articles by Appelbaum, P. C.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»