Biochemical characterization of penicillin-resistant and -sensitive penicillin-binding protein 2x transpeptidase activities of Streptococcus pneumoniae and mechanistic implications in bacterial resistance to beta-lactam antibiotics.

To understand the biochemical basis of resistance of bacteria to beta-lactam antibiotics, we purified a penicillin-resistant penicillin-binding protein 2x (R-PBP2x) and a penicillin-sensitive PBP2x (S-PBP2x) enzyme of Streptococcus pneumoniae and characterized their transpeptidase activities, using a thioester analog of stem peptides as a substrate. A comparison of the k(cat)/Km values for the two purified enzymes (3,400 M(-1) s(-1) for S-PBP2x and 11.2 M(-1) s(-1) for R-PBP2x) suggests that they are significantly different kinetically. Implications of this finding are discussed. We also found that the two purified enzymes did not possess a detectable level of beta-lactam hydrolytic activity. Finally, we show that the expression levels of both PBP2x enzymes were similar during different growth phases.

Cloning and characterization of femA and femB from Staphylococcus epidermidis.

A DNA fragment was identified and cloned from Staphylococcus epidermidis (Se) using femA from S. aureus (Sa) as a heterologous hybridization probe. DNA sequence analysis of a portion of this clone revealed two complete ORFs highly related to femA and femB of Sa. The genomic arrangement of the Se femA/B complex was nearly identical to that observed in Sa. Intra- and interspecies relatedness of these genes and conservation of genomic organization were consistent with gene duplication of one of these genes in an ancestral organism. Recombinant FEMA, produced in Escherichia coli (Ec), was purified to near homogeneity. Identity of the purified protein was verified by N-terminal amino acid (aa) sequence analysis.

Semisynthetic glycopeptide antibiotics derived from LY264826 active against vancomycin-resistant enterococci.

Certain derivatives of the glycopeptide antibiotic LY264826 with N-alkyl-linked substitutions on the epivancosamine sugar are active against glycopeptide-resistant enterococci. Six compounds representing our most active series were evaluated for activity against antibiotic-resistant, gram-positive pathogens. For Enterococcus faecium and E. faecalis resistant to both vancomycin and teicoplanin, the MICs of the six semisynthetic compounds for 90% of the strains tested were 1 to 4 micrograms/ml, compared with 2,048 micrograms/ml for vancomycin and 256 micrograms/ml for LY264826. For E. faecium and E. faecalis resistant to vancomycin but not teicoplanin, the MICs were 0.016 to 1 micrograms/ml, compared with 64 to 1,024 micrograms/ml for vancomycin. The compounds were highly active against vancomycin-susceptible enterococci and against E. gallinarum and E. casseliflavus and showed some activity against isolates of highly vancomycin-resistant leuconostocs and pediococci. The MICs for 90% of the strains of methicillin-resistant Staphylococcus aureus tested were typically 0.25 to 1 micrograms/ml, compared with 1 microgram/ml for vancomycin. Against methicillin-resistant S. epidermidis MICs ranged from 0.25 to 2 micrograms/ml, compared with 1 to 4 micrograms/ml for vancomycin and 4 to 16 micrograms/ml for teicoplanin. The spectrum of these new compounds included activity against teicoplanin-resistant, coagulase-negative staphylococci. The compounds exhibited exceptional potency against pathogenic streptococci, with MICs of < or = 0.008 microgram/ml against Streptococcus pneumoniae, including penicillin-resistant isolates. In in vivo studies with a mouse infection model, the median effective doses against a challenge by S. aureus, S. pneumoniae, or S. pyogenes were typically 4 to 20 times lower than those of vancomycin. Overall, these new glycopeptides, such as LY307599 and LY333328, show promise for use as agents against resistant enterococci, methicillin-resistant S. aureus, and penicillin-resistant pneumococci.

Activities of the semisynthetic glycopeptide LY191145 against vancomycin-resistant enterococci and other gram-positive bacteria.

LY191145 is the prototype of a series of compounds with activities against vancomycin-resistant enterococci derived by modification of the glycopeptide antibiotic LY264826. LY191145 had MICs for vancomycin- and teicoplanin-resistant enterococci of < or = 4 micrograms/ml for 50% of isolates and < or = 16 micrograms/ml for 90% of isolates. Its MICs for vancomycin-resistant, teicoplanin-susceptible enterococci were 1 to 8 micrograms/ml. LY191145 retains the potent activities of its parent compound against staphylococci and streptococci. In vivo studies in a mouse infection model confirmed these activities. This compound indicates the potential of semisynthetic glycopeptides as agents against antibiotic-resistant gram-positive bacteria.

Site-directed mutagenesis of the mecA gene from a methicillin-resistant strain of Staphylococcus aureus.

The mecA-27r gene from Staphylococcus aureus 27r encodes penicillin-binding protein 2a (PBP2a-27r), which causes this strain to be methicillin resistant. Removal or replacement of the N-terminal transmembrane domain had no effect on binding of penicillin, but removal of portions of the putative transglycosylase domain (144, 245, or 341 amino acids after the transmembrane region) destroyed penicillin-binding activity. The SXXK, SXN, and KSG motifs, present in all penicillin-interacting enzymes, were found in the expected linear spatial arrangement within the putative transpeptidase region of PBP2a-27r. Alterations of amino acids in all three of these motifs resulted in elimination of penicillin-binding activity, confirming their roles in the interaction with penicillin.

In-vitro synergy and mechanism of interaction between vancomycin and ciprofloxacin against enterococcal isolates.

In-vitro synergy between vancomycin and ciprofloxacin against 44 enterococcal isolates was studied. Synergy occurred in chequerboard MIC determinations with six Enterococcus faecium strains resistant to both vancomycin and ciprofloxacin. The combination was additive for strains susceptible to one or both antibiotics. Time-kill studies involving selected strains with different susceptibility patterns confirmed the chequerboard results. The effect of ciprofloxacin on the induction of vancomycin resistance was compared in two vancomycin-resistant strains of E. faecium. Sub-inhibitory concentrations of ciprofloxacin prevented induction of vancomycin resistance in a ciprofloxacin-resistant strain, but not in a ciprofloxacin-susceptible strain. Membranes isolated from vancomycin-resistant ciprofloxacin-resistant cultures grown with vancomycin and ciprofloxacin at < or = 8 mg/L (0.125 x MIC) expressed a 39.5-kDa membrane protein involved in the expression of vancomycin resistance, but the protein was not detected in membranes from cultures grown in ciprofloxacin 16 mg/L. These findings indicated that a vancomycin-ciprofloxacin combination can be synergic against enterococci resistant to both vancomycin and ciprofloxacin, but would be unlikely to offer any advantage in the treatment of enterococcal infections because of the high concentrations required.

Detection of methicillin-resistant staphylococci by using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based test was developed for the detection of mecA in staphylococci. To facilitate this process, a rapid cell lysis procedure was established for the release of DNA from staphylococcal strains. Primers based on the DNA sequence of the mecA gene from Staphylococcus aureus were used in PCRs to screen for the presence of this gene in a total of 98 staphylococcal isolates. Fifty-one isolates were mecA positive (17 S. aureus strains and 34 coagulase-negative staphylococci including S. epidermidis, S. haemolyticus, and S. simulans). Results obtained with PCRs were generally consistent with those of standard microbiological assays. PCRs designed to detect the femA gene (factor essential for methicillin resistance) revealed the presence of the gene in all S. aureus strains examined regardless of the susceptibility profiles of the strains to methicillin. In contrast, femA could not be detected in coagulase-negative staphylococci by PCR with the same primers. Low-stringency hybridization suggested the presence of a gene structurally related to femA in S. epidermidis and other coagulase-negative staphylococci examined.