The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired -lactam resistance was investigated by constructing isogenic strains of Enterococcus faecium producing different PBP 5. The pbp5 genes from three E. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faecium D344 highly susceptible to ampicillin due to deletion of pbp5 (MIC, 0.03 g/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 g/ml), D344 (MIC, 24 g/ml), and H80721 (MIC, 512 g/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 g/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.Enterococcus faecium is intrinsically resistant to moderate levels of ampicillin by production of the low-affinity penicillinbinding protein 5 (PBP 5). Bacterial growth occurs at -lactam concentrations sufficient to inactivate all the other PBPs, suggesting that PBP5 is the only transpeptidase required for peptidoglycan synthesis under such conditions (4,20). Acquired resistance to higher levels of ampicillin in clinical isolates of E. faecium has been associated with increased production of PBP 5 or decreased affinity for the -lactam antibiotics (7,8,10,13,18,20,21). The latter mechanism was inferred from comparison of the pbp5 genes from clinical isolates in which amino acid substitutions at specific positions near or in the conserved motifs of the PBP module were associated with decreased interaction with -lactams and expression of resistance (8,13,18,21). However, the role of the PBP 5 in the level of resistance was not rigorously established since isogenic strains were not constructed. In the present study, pbp5 genes from E. faecium clinical isolates expressing various levels of ampicillin resistance were cloned into a shuttle vector and introduced into E. faecium D344S, a spontaneous mutant in which the chromosomal pbp5 locus is deleted. Expression of the different pbp5 genes in this host resulted in similar low levels of ampicillin resistance, indicating that alterations of PBP 5 alone do not account for acquired high-level -lactam resist...
We report a structural and transcriptional analysis of the pbp5 region of Enterococcus faecium C68. pbp5 exists within a larger operon that includes upstream open reading frames (ORFs) corresponding to previously reported psr (penicillin-binding protein synthesis repressor) and ftsW (whose product is a transmembrane protein that interacts with PBP3 in Escherichia coli septum formation) genes. Hybridization of mRNA from C68, CV133, and four ampicillin-resistant CV133 mutants revealed four distinct transcripts from this region, consisting of (i) E. faecium ftsW (ftsW Efm ) alone; (ii) psr and pbp5; (iii) pbp5 alone; and (iv) ftsW Efm , psr, and pbp5. Quantities of the different transcripts varied between strains and did not always correlate with quantities of PBP5 or levels of ampicillin resistance. Since the psr of C68 is presumably nonfunctional due to an insertion of an extra nucleotide in the codon for the 44th amino acid, the region extending from the ftsW Efm promoter through the pbp5 gene of C68 was cloned in E. coli to facilitate mutagenesis. The psr ORF was regenerated using site-directed mutagenesis and introduced into E. faecium D344-SRF on conjugative shuttle vector pTCV-lac (pCWR558 [psr ORF interrupted]; pCWR583 [psr ORF intact]). Ampicillin MICs for both D344-SRF-(pCWR558) and D344-SRF(pCWR583) were 64 g/ml. Quantities of pbp5 transcript and protein were similar in strains containing either construct regardless of whether they were grown in the presence or absence of ampicillin, arguing against a role for PSR as a repressor of pbp5 transcription. However, quantities of psr transcript were increased in D344-SRF(pCWR583) compared to D344-SRF(pCWR558), especially after growth in ampicillin; suggesting that PSR acts in some manner to activate its own transcription.Penicillin resistance in Enterococcus faecium is associated with production of low-affinity penicillin-binding protein PBP5. The presence of this penicillin-binding protein (PBP) in virtually all clinical E. faecium strains that have been investigated (including those susceptible to clinically achievable levels of penicillin [L. B. Rice, unpublished data]) suggests that it is intrinsic to this species, rather than an acquired gene. Supportive evidence for the role of PBP5 in penicillin resistance is derived from experiments indicating that PBP5-expressing cells replicate when incubated with penicillin at concentrations sufficient to saturate all of the other PBPs, as well as from studies demonstrating that E. faecium strains lacking PBP5 are highly susceptible to penicillin (10-12, 25, 26).Early studies on Enterococcus hirae 9790 (which until 1985 was considered to be a type strain for Enterococus faecalis [8,16]) reported that elevated levels of penicillin resistance (to ca. 64 g/ml) were associated with increased quantities of detectable PBP5. Increased PBP5 production in one resistant mutant (R40) was associated with deletion of the N-terminal portion and some upstream DNA of an open reading frame (ORF) located ca. 1 kb upstream of the pbp5...
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.
Topoisomerase IV, a C 2 E 2 tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases, which modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (59). Topoisomerase IV forms a C 2 E 2 tetramer involved in segregation of the chromosome at cell division (1, 30, 59). The ParC subunit contains the site of topoisomerization catalyzing the double-stranded DNA break (30, 53), while the ParE subunit catalyzes the hydrolysis of ATP, providing the free energy necessary for these reactions (2, 6, 11). The ParE and ParC subunits share extensive sequence homology with, respectively, the GyrB and GyrA subunits of the DNA gyrase A 2 B 2 tetramer, which catalyzes negative DNA supercoiling during the initiation and elongation processes of DNA replication (16,53).Both topoisomerases are the targets of antibacterial molecules, such as the quinolones and the coumarins. The coumarins inhibit supercoiling and enzyme turnover by preventing the binding and hydrolysis of ATP (45). The quinolones form a ternary complex with the topoisomerases in the presence of DNA, resulting in lethal double-stranded DNA breaks (11). Enzymatic studies and binding assays have also shown that the quinolones can form a complex with the topoisomerases before binding DNA (15, 29); however, some binding data indicate the occurrence of a specific and higher level of binding to the enzyme-DNA complex rather than to the enzyme alone (43,56). These families of molecules, in particular, the fluoroquinolones (FQs), are under continuous development as resistance to antibiotics in pathogenic bacteria has dramatically increased during the last decade (20,22,49).
Against penicillin-susceptible pneumococci, the activity of sanfetrinem was similar to those of penicillin, amoxicillin, cefotaxime, imipenem, and meropenem, while against penicillin-resistant strains, sanfetrinem and the carbapenems exhibited superior activity (MICs at which 90% of strains are inhibited, ≤1 μg/ml). PBP 1a in the penicillin-susceptible strain and PBP 1a and PBP 2b in the more resistant isolates seemed to be the essential penicillin-binding proteins for imipenem and sanfetrinem.
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