Mechanisms of Penicillin Resistance in Streptococcus pneumoniae: Targets, Gene Transfer, and Mutations

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Dalia Denapaite, Fang Chi, Patrick Maurer, Oliver Nolte, and Regine Hakenbeck
Abstract Penicillin-binding proteins, PBPs, are crucial enzymes important for the biosynthesis of murein, the typical prokaryotic macromolecule. They represent the targets for beta-lactam antibiotics and are thus involved in the evolution of penicillin resistance in Streptococcus pneumoniae. Whereas distinct point mutations in individual pbp genes occur in laboratory mutants selected for resistance, gene transfer events play an additional role for the emergence and spread in clinical isolates. Moreover, apparent PBP independent resistance mechanisms have been described. In this chapter, alterations in PBPs of resistant strains on the molecular level will be discussed and the different pathways now identified during the development of resistance to different beta-lactams will be summarized. Introduction Penicillin resistance in Streptococcus pneumoniae is a complex multistep process that involves alterations of penicillin target enzymes, the penicillin binding proteins (PBPs). In contrast to staphylococci or enterococci, beta-lactamase production has never been observed in the pneumococcus. In addition, non-pbp genes contribute to resistance. This phenomenon was observed for the first time in laboratory mutants, but meanwhile there is increasing evidence that also in clinical isolates there is more to resistance than only mutations in pbp genes. There is at least one main difference between beta-lactam resistant laboratory mutants and clinical isolates. Whereas point mutations are selected in the laboratory that can easily be identified in the genes (once the gene affected has been characterized), clinical isolates incorporate ready-made pbp genes via gene transfer followed by recombination into the chromosomal gene. These resistant genes probably have evolved from sensitive closely related oral streptococci that contain genes closely related to the sequences found in the mosaic blocks of resistant S. pneumoniae pbp genes. These mosaic blocks therefore contain all the alterations that are due to the foreign origin of the gene fragment itself in addition to the mutations relevant for resistance that are therefore difficult to identify. The order to pbp changes during the evolution of resistance is largely dependent on the selective beta-lactam as has been shown by detailed analyses of laboratory mutants. Details of the selective forces operating in the host environment in vivo, however, are less clear. In the following article,

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the role of PBPs in resistant clinical isolates and laboratory mutants will be described, and mutations relevant for resistance in pbp and non-pbp genes will be reviewed. Penicillin-binding proteins in S. pneumoniae The PBPs are multidomain proteins with extracytoplasmic domains that function during late steps of murein biosynthesis. They all contain a penicillin-binding domain with three conserved amino acid motifs: SXXK with the active site serine responsible for the covalent linkage to the beta-lactam antibiotic, an SXN and a KS(T)G box. This domain is involved in the crucial penicillin-sensitive step of murein biosynthesis, the transpeptidation of muropeptides, and only here mutations important for resistance development have been identified. The mutations result in a decreased affinity to beta-lactams; hence a higher concentration of the antibiotic is needed for inhibition of the PBP function and consequently for in vivo growth inhibition and lysis.

According to their overall domain structure, PBPs are classified as high molecular weight (hmw) PBPs of class A possessing an N-terminal glycosyltransferase domain whose activity can be inhibited by the antibiotic moenomycin, hmw PBPs of class B with an N-terminal domain of unknown function, and low molecular weight (lmw) PBPs that act mainly as D,D-carboxypeptidases (for review see Goffin and Ghuysen, 2002). The function of their C-terminal domains is not known. The hmw PBPs are integrated into the membrane via a short N-terminal hydrophobic segment, whereas lmw PBPs contain an amphiphilic helix at the C-terminus that functions as membrane attachment domain. Due to their ability to covalently bind beta-lactams PBP can easily be visualized after incubation with radioactive or fluorescent antibiotic and separation on SDS polyacrylamide gels as described in the method section of this book. In each organism, PBPs are named according to their apparent molecular weight in decreasing order. Thus, S. pneumoniae contains six PBPs named PBP1a, 1b, 2x, 2a, 2b and 3. This somewhat mysterious nomenclature is purely historical. The first gels run in the late 70s resolved three PBP bands hence named PBP1, 2 and 3, but soon improvement of the SDS gel technology resulted in resolution into the two pairs PBP1a + 1b and 2a + 2b. Only years later during purification and biochemical characterization of the PBPs, a sixth PBP was recognized and named PBP2x. It is curious that it is this last PBP that has served as a model PBP for structural and functional analyses for our understanding of the evolution of penicillin resistance. PBP function Since deletion mutants neither in the two class B hmw PBP2x and PBP2b could be isolated they are believed to be the two essential PBPs of S. pneumoniae (Kell et al., 1993). The class A hmw PBP1a, 1b and 2a can be individually deleted, and double mutants have also be obtained except for the pair pbp1a/pbp2a (Paik et al., 1999; Hoskins et al., 1999). The pbp2a mutant showed a higher susceptibility to moenomycin (which inhibits the transglycosylase activity in E. coli PBP1b) this protein was therefore suggested to be the main transglycosylase in the pneumococcus (Paik et al., 1999; Hoskins et al., 1999). Isolated PBP2a derivatives contain indeed transglycosylase activity in vitro (Di Guilmi et al., 2003b). The class A PBP double mutants showed severe effects in cell morphology being unable to synthesize regular division septa, and an increased sensitivity to stationary phase lysis was also

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observed (Paik et al., 1999). A transglycosylase (glycosyltransferase) activity has indirectly been documented for PBP1b where a GT domain dependent binding to lipid II which could be inhibited by moenomycin has been demonstrated (Di Guilmi et al., 2003a). The lmw PBP3 shown to be a D,D-carboxypeptidase in vitro (Hakenbeck and Kohiyama, 1982) is dispensable, but deletion mutants grow poorly and show highly aberrant division septa, a thickened cell wall and shedding of wall material into the growth medium (Schuster et al., 1990), and the murein composition was also highly altered, suggesting that the D,D-carboxypeptidase activity is an important function for cell division (Severin et al., 1992). Whereas PBP3 localizes over the entire surface (Hakenbeck et al., 1993), it appears to be absent at the division septum and the rings formed by hmw PBPs and that of FtsZ are no longer colocalized (Morlot et al., 2004). PBPs and beta-lactam resistance: lysis versus tolerance In agreement with the notion that PBP2x and PBP2b are the only essential PBPs in S. pneumoniae both proteins are primary targets, i.e. alterations in PBP2x or PBP2b alone confer resistance. In contrast, mutations in other PBPs do not result in a detectable susceptibility decrease if they are not accompanied by PBP2x and/or PBP2b alterations. The contribution of any other PBP alteration to resistance requires changes in either one, or both, essential PBPs. Resistance mediated by mutations in either PBP2b or PBP2x never reaches high levels: e.g. a low affinity PBP2x results in cefotaxime resistance (which selects well for PBP2x mutations) of MIC values around 0.040.2 g/ml compared to 0.02 g/ml in sensitive strains (Laible and Hakenbeck, 1991; Krau et al., 1996; Sifaoui et al., 1996), whereas piperacillin selects primarily mutations in PBP2b resulting in only a twofold resistance increase from 0.04 to 0.060.08 g/ml piperacillin (Hakenbeck et al., 1994; Grebe and Hakenbeck, 1996). Since PBP2b does not interact with cefotaxime over a wide concentration range (or other third generation cephalosporins and aztreonam which has a

similar side chain), it is not a target for this class of compounds (Hakenbeck et al., 1987). Most remarkably, cefotaxime treatment does not result in rapid lysis and cells are also killed at a much lower rate compared to treatment with penicillin antibiotics that generally induce lysis rapidly, i.e. cefotaxime induces a tolerant response (Hakenbeck et al., 1987). In other words, inhibition of PBP2b appears to be a prerequisite for the lytic response. High level resistant clinical isolates usually contain a low affinity PBP2b, and the early observation that penicillin-resistant strains appear to be tolerant (Liu and Tomasz, 1985) has been associated with the presence of a low affinity PBP2b (Hakenbeck et al., 1987). S. pneumoniae strains that contain only a low affinity PBP2b have indeed been shown to display a tolerant response for penicillin antibiotics (Grebe and Hakenbeck, 1996; Reichmann et al., 1997). This scenario has important implications. First, there is one PBP target less for cefotaxime and related beta-lactams, and therefore high level resistance requires only changes in pbp2x and pbp1a (Muz et al., 1992; Dowson et al., 1994; Barcus et al., 1995; Hakenbeck et al., 1998). The appearance of high level cefotaxime resistant clones with changes in only PBP2x and PBP1a in the USA and South Africa supports this notion (Coffey et al., 1995; McDougal et al., 1995; Smith et al., 2001). Second, it suggests that cells with a low affinity PBP2b are better survivors, and thus might have an advantage over wild type cells even in vivo without any selection pressure.

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All PBPs have been implicated in high level resistance (Figure 11.1). An altered PBP1a has been recognized already in the first reports describing high level penicillin resistant clinical isolates (Hakenbeck et al., 1980; Percheson et al., 1980). Its contribution to resistance has been proven by transformation of pbp1a genes encoding low affinity mosaic PBP1a into a pbp2x background (using cefotaxime for selection) or a recipient strain containing both, an altered pbp2b as well as pbp2x gene in case of penicillin selection (Muz et al., 1992). PBP2a plays a role as a resistance determinant in cefotaxime resistant laboratory mutants after the introduction of a low affinity PBP2a (Laible and Hakenbeck, 1987). Some clinical isolates contain a low affinity PBP2a which has also been observed in beta-lactam resistant S. pneumoniae transformants obtained with DNA from resistant S. mitis or S. oralis, suggesting that it also plays a role for resistance in the clinical setting (Reichmann et al., 1997; Hakenbeck et al., 1998; du Plessis et al., 2002). However, an altered pbp2a gene could not be transformed with beta-lactam selection (du Plessis et al., 2000). A low affinity PBP1b has been described only in resistant S. pneumoniae transformants obtained with donor DNA from a high level resistant S. mitis (Hakenbeck et al., 1998), whereas sequence comparison of high level resistant strains did not reveal PBP1b changes (Hakenbeck et al., 1998). Alterations in PBP3 have been described in clinical isolates as a clonal property and are probably independent of their resistance phenotype, but a mutation resulting in a low affinity variant which contributed to cefotaxime resistance has been described in a resistant laboratory mutant (Krau and Hakenbeck, 1997); also the amount of PBP3 has been linked to alterations in cefotaxime resistance (Selakovitch-Chenu et al., 1993).
Figure 11.1 Penicillin binding proteins in beta-lactam resistant Streptococcus pneumoniae. PBPs are schematically shown as bars. s: sensitive strains; Cef: cefotaxime resistant mutants; Pip: piperacillin resistant mutants; T (S. mitis): S. pneumoniae transformants obtained with DNA from high level resistant oral streptococci. The line representation of PBP1a in the Pip mutants documents less protein. Arrows indicate the PBPs that are the first PBPs altered in successively isolated mutants. White: mutated or altered PBPs in high level resistant mutants or transformants compared to sensitive strains.

PBP 1a 1b 2a 2b 2x 3
s Cef Pip clinical isolates

T(S. mitis)

Hakenbeck, Fig. 1

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Gene transfer and the evolution of mosaic genes in clinical isolates The pbp genes in resistant isolates encoding low affinity PBPs are highly variable due to the presence of sequence blocks that differ approximately 20% compared to corresponding sequences in sensitive strains. Mosaic genes have been described in the three pbp genes involved in high level resistance pbp1a (Martin et al., 1992), pbp2b (Dowson et al., 1989) and pbp2x (Laible et al., 1991). The altered amino acid sequences in the resistant PBPs result in electrophoretic mobility shifts most pronounced in PBP1a (Laible et al., 1991) although their calculated molecular weight is almost identical. Since these changes are generally clone specific the PBP profiles on SDS PAGE in combination with antibody reactivity pattern can be used as clonal markers (Hakenbeck et al., 1991). Restriction fragment length polymorphism (RFLP) of pbp genes has also been used as a DNA based screen to establish clonal relatedness and to identify horizontal gene transfer of mosaic pbp genes between different clones of S. pneumoniae (Coffey et al., 1991; Muz et al., 1991). Although these methods are useful for screening a large number of isolates, small changes in the size of the mosaic blocks and point mutations in pbp genes that are important for the deduction of the evolutionary history might be missed by these analyses. Alterations in pbp2a genes also occur occasionally in some high level resistant strains with up to 6% differences (Hakenbeck et al., 1998; du Plessis et al., 2000; Sanbongi et al., 2004) (and own unpublished results), and a highly altered pbp1b gene has only been observed in transformants obtained with DNA of a high level penicillin resistant S. mitis (Hakenbeck et al., 1998; Sanbongi et al., 2004). Sequences highly related to mosaic pbp blocks of resistant S. pneumoniae have been detected in pbp genes from resistant commensal streptococci, showing that similar to Neisseria spp. interspecies gene transfer involving these resistance determinants occurs (Dowson et al., 1990; Reichmann et al., 1997). Sequences related to resistant mosaic pbp2x (Sibold et al., 1994) and pbp2b (Dowson et al., 1993) of S. pneumoniae were also found in susceptible S. mitis strains, and it is therefore likely that low affinity PBPs have evolved in commensal streptococci prior to interspecies gene transfer into the pneumococcal population (Figure 11.2). Transformation of a pbp2x gene from a sensitive S. mitis into a sensitive S. pneumoniae strain was possible, showing that other evolutionary pathways cannot be excluded (Sibold et al., 1994). One major class of pbp2x genes exists that has apparently spread into many different S. pneumoniae clones and occurs in resistant S. mitis and S. oralis strains as well (Reichmann et al., 1997). In addition, a surprisingly large number of pbp2x variants exists, and the origin of this variability is not known. Recombination events resulting in altered pbp genes can occur within the gene or in flanking regions. It has been observed in PBP2x from resistant isolates that the block structure reflects the domain structure of the protein in many cases probably due to a high selective pressure on the function of the protein (Sibold et al., 1994). Alterations in adjacent genes include the ddl gene upstream of pbp2b (Enright and Spratt, 2004) and the ftsL gene upstream of pbp2x (own unpublished results). Since the capsular gene locus is flanked by the pbp1a and pbp2x genes, intraspecies transformation of resistance can result in capsular switching as well (Trzcinski et al., 2006) as has been shown to occur in natural populations

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(Coffey et al., 1998; Coffey et al., 1999). pbp genes that are located at a great distance on the chromosome such as pbp2b and pbp2x, or pbp2a and pbp2x, can be affected within one transformation step as has been shown with chromosomal DNA from resistant S. mitis, suggesting that other parts of the chromosome could be altered as well during interspecies transformation events (Reichmann et al., 1997; Hakenbeck et al., 1998).

Mutations in pbp genes associated with resistance and PBP structures Mutational analysis of laboratory mutants revealed the complexity of resistance development. Low affinity variants of PBP2x, for example, can easily be selected with cefotaxime. Already after single step selection different mutations occur (Sifaoui et al., 1996; Negri et al., 2002), and six independent mutants obtained after a multistep selection procedure resulted in six completely distinct, multiply mutated proteins with up to four point mutations (Laible and Hakenbeck, 1991; Krau et al., 1996). It is remarkable that most mutations thus identified did not map close to the active site except for the two mutations adjacent to the K547SG box T550A and Q552E, and H394Y next to the S395SN box. The mutation T550A is remarkable since it confers high level cefotaxime resistance and simultaneously hypersensitivity to penicillins in laboratory mutants (Laible and Hakenbeck, 1987; Laible and Hakenbeck, 1991); occasionally it occurs in high level cephalosporin resistant clinical isolate (Coffey et al., 1995). A T550G mutation which includes two base changes within that codon increases the cefotaxime resistance even further (Grebe and Hakenbeck, 1996). The H394Y change has also been identified in PBP2x of clinical isolates (Nagai et al., 2002); the effect of H394L that occurs frequently in clinical isolates has not been experimentally investigated. Generally, mutations in pbps of resistant clinical isolates are not easily recognizable due to the mosaic structure of the genes. Some PBP2x mutations have been verified as resistance determinants by a combination of mutagenesis and biochemical characterization
resistant laboratory mutant clinical isolate susceptible S. pneumoniae susceptible S. mitis resistant resistant (1) resistant (2)
Hakenbeck, Fig. 2

Figure 11.2 Evolution of mosaic PBP genes. White: pbp genes of penicillin susceptible S. pneumoniae; black: pbp genes of oral streptococci. Lines indicate mutations that confer penicillin resistance.

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of the protein. Among these are alterations close to conserved boxes at positions T338 (to A,G,P or S), M339F, and Q552E (Mouz et al., 1998; Chesnel et al., 2003; Pernot et al., 2004; Smith and Klugman, 2005). Kinetic parameters of isolated soluble PBP2x derivatives confirmed that the overall binding efficiency of a resistant PBP2x is much slower than that for sensitive PBP2x (k2/Kd values of 100 000 to 200 000 M1s1 for sensitive PBP2x compared to 85 M1s1 and lower for resistant PBP2x containing multiple alterations) (Figure 11.3; Jamin et al., 1992; Lu et al., 2001; Carapito et al., 2006). Many alterations that are found in the mosaic blocks are probably irrelevant for resistance since they occur also in PBPs of sensitive streptococci (Dowson et al., 1993; Sibold et al., 1994) (and own unpublished results). Among these alterations are some that have been suggested to contribute to resistance. For example, in PBP2x Q447M, S449A and N514H are close to the active site and have therefore be proposed contribute to structural alterations of the active site in resistant strains (Dessen et al., 2001; Pernot et al., 2004). Interestingly, the R384G change that also occurs in a sensitive S. mitis (own unpublished results) influences the susceptibility (Smith and Klugman, 2005; Carapito et al., 2006), and altered the acylation efficiency of the protein (Carapito et al., 2006). Changes close to the conserved boxes have been shown to be relevant for other resistant PBPs as well. In PBP2b, G660D at the C-terminal end of the protein, G617A within the K615SG motif (Hakenbeck et al., 1994), and T446A close to the S443SN box (Grebe and Hakenbeck, 1996) have been selected with piperacillin in the laboratory. The T446A change occurs in many resistant clinical isolates (Dowson et al., 1993), and changes at the C-terminal end of PBP2b have also been implicated in the resistance process of clinical isolates (Dowson et al., 1989; Chi, 2004). In resistant PBP1a of clinical isolates, the changes T371A or T371S close to the active site S370 occur frequently and contribute to resistance (Smith and Klugman, 1998; Asahi et al., 1999), and residues 574 to 577 are also important (Smith and Klugman, 1998; Smith and Klugman, 2003). Again, positions that are also altered in pbp1a genes of sensitive S. mitis (own unpublished results) have been

described as potentially important for the resistance process for structural reasons ( Job et al., 2003); their actual impact on resistance and interaction with beta-lactams with the protein in vivo is not yet known. Deletion of pbp2a in laboratory mutants also increases the resistance (van der Linden and Hakenbeck, unpublished results), but mutations in clinical
Figure 11.3 Mutations in PBP2x of laboratory mutants. The region covers the transpeptidase domain of PBP2x only (amino acids 266 to 616). The three active site boxes are indicated on top. The amino acids of the sensitive S. pneumoniae R6 strain are shown. Positions that have been identified also in mosaic pbp2x of clinical isolates are marked (*).
289 394 403 422 426 458 512 526 550 596 600

S337TMK S395SN K547SG (266) (616) M H* L GR Q R T T* SG LG laboratory mutants Q*552

597 601

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isolates have not yet been correlated with resistance although the T411A mutation in the active site S410TIK motif has been found in high level resistant strains and transformants (Hakenbeck et al., 1998; Sanbongi et al., 2004). In pbp3 of a cefotaxime resistant laboratory mutant the mutation T242I close to the K239TG box was identified (Krau and Hakenbeck, 1997). The impact of interpeptide crosslinkage on resistance In Gram positive bacteria, muropeptides are often crosslinked via a short interpeptide bridge which in S. pneumoniae consists of l-Ala-l-Ala and/or l-Ala-l-Ser (Garcia-Bustos and Tomasz, 1990). The transpeptidation with the muropeptides is catalyzed by gene products named FibAB (Weber et al., 2000) or MurMN (Filipe and Tomasz, 2000). They belong to the family of Fem peptidyltransferases first described in methicillin resistant Staphylococcus aureus, where disruption of the fem genes (factor essential for methicillin resistance) resulted in a methicillin sensitive phenotype (for review, see Rohrer and Berger-Bchi, 2003). Similarly, disruption of the fibAB/murMN operon in high level resistant S. pneumoniae caused a complete collapse of penicillin resistance and to a reduction in crosslinked muropeptides (Weber et al., 2000; Filipe and Tomasz, 2000). Since then the function of these gene products has been under investigation, and the relationship to resistance is still not clear. Some penicillin resistant clinical isolates contain altered murein and altered murMN alleles (Garcia-Bustos and Tomasz, 1990), but other resistant clones do not (Enright and Spratt, 2004; Cafini et al., 2006). In transformation studies involving a high level resistant Hungarian isolate transformation of full resistance involved an altered murM allele in addition to the pbp genes (Smith and Klugman, 2001), but PBP mediated resistance and altered muropeptide composition could be separated in transformation experiments (Severin et al., 1996; Hakenbeck et al., 1998). PBPs catalyze the crosslinking between two muropeptides, and thus must use the substrates whose synthesis involves the activity of the Fem proteins. Since PBPs of resistant isolates are highly variable, they may accept different muropeptides as substrates depending on the mutations that affect the active site in a more or less stringent manner, and it has been suggested that PBP2x may be responsible for the penicillin sensitivity in fib mutants (Weber et al., 2000). It is also possible that cell wall associated proteins containing the LPXTG motif that are covalently bound to the interpeptide bridges via the sortase play a still unknown role for the resistance phenotype. Biochemical and structural investigations will be needed to solve these problems. Non-PBP mutations and beta-lactam resistance Although transformation of pbp genes from resistant clinical isolates frequently results in transformants that are apparently as resistant as the donor strains, exceptions have been noted. In some cases, the murM gene that is also altered in some resistant strains accounted for this discrepancy as mentioned above whereas in other cases the reason for this is not clear (Smith and Klugman, 2000; Chesnel et al., 2005). In laboratory mutants it was noted for some time that mutations in non-pbp genes also occur during the selection with betalactams. In piperacillin resistant mutants, a putative membrane associated glycosyltransferase CpoA was affected that was suggested to play a role in teichoic acid biosynthesis (Grebe et al., 1997); its function as a lipid glycosyl-transferase has recently been verified

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biochemically (Edman et al., 2003). The cpoA mutants were slightly less susceptible to piperacillin and contained less PBP1a, but an explanation on the molecular level of this complex phenotype is not known. All cefotaxime resistant mutants contained mutations in the histidine protein kinase CiaH, with every mutant containing a different ciaH allele (Zhner et al., 2002). The CiaRH two component system apparently is required during cell wall stress: deletion mutants in ciaR are unusually lysis prone and hypersensitive to a wide variety of early and late cell wall inhibitors, whereas mutants with an activated CiaRH system were highly resistant to many different lysis inducing conditions (Mascher et al., 2006). Moreover, deletion of the response regulator in mutants containing a low affinity PBP2x showed severe growth defects and lyzed rapidly. This defect was especially marked with PBP2x from laboratory mutants containing the T550A change, whereas it was less pronounced in the presence of resistant PBP2x from clinical isolates. This strongly suggests that PBP2x mutations are functionally not neutral, and that this defect can be balanced by a functional CiaRH system. Mutations in CiaH have not yet been observed in clinical isolates. Since CiaH mutations have a complex phenotype and affect the genetic competence as well (Mascher et al., 2003), it might be required in the in vivo situation in agreement with the finding that CiaRH mutants are attenuated in mouse models (Throup et al., 2000; Sebert et al., 2002; Marra et al., 2002). In summary, the evolution of resistance in S. pneumoniae represents a highly complicated scenario, involving target proteins such as PBPs and non-PBP components as well. Laboratory experiments clearly documented that the kind of mutations and genes selected during resistance development varies enormously depending on the selective compound. Moreover, the complex mosaic structures found in resistant clinical isolates suggests that many different ways for the restructuring of PBPs exist, similar to what has been found in laboratory mutants. Acknowledgment This work was supported by the DFG (Ha 1011/9-1) the BMBF (PTJ-BIO/0313134), the EU (LSHM-CT-2003-503413 and -503335), and the Schwerpunkt Biotechnologie at the University of Kaiserslautern. References
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