Structural Insights into the TLA-3 Extended-Spectrum ß-Lactamase and Its Inhibition by Avibactam and OP0595.

The development of effective inhibitors that block extended-spectrum ß-lactamases (ESBLs) and restore the action of ß-lactams represents an effective strategy against ESBL-producing Enterobacteriaceae We evaluated the inhibitory effects of the diazabicyclooctanes avibactam and OP0595 against TLA-3, an ESBL that we identified previously. Avibactam and OP0595 inhibited TLA-3 with apparent inhibitor constants (Kiapp) of 1.71 ± 0.10 and 1.49 ± 0.05 µM, respectively, and could restore susceptibility to cephalosporins in the TLA-3-producing Escherichia coli strain. The value of the second-order acylation rate constant (k2/K, where k2 is the acylation rate constant and K is the equilibrium constant) of avibactam [(3.25 ± 0.03) × 103 M-1 · s-1] was closer to that of class C and D ß-lactamases (k2/K, <104 M-1 · s-1) than that of class A ß-lactamases (k2/K, >104 M-1 · s-1). In addition, we determined the structure of TLA-3 and that of TLA-3 complexed with avibactam or OP0595 at resolutions of 1.6, 1.6, and 2.0 Å, respectively. TLA-3 contains an inverted Ω loop and an extended loop between the ß5 and ß6 strands (insertion after Ser237), which appear only in PER-type class A ß-lactamases. These structures might favor the accommodation of cephalosporins harboring bulky R1 side chains. TLA-3 presented a high catalytic efficiency (kcat/Km ) against cephalosporins, including cephalothin, cefuroxime, and cefotaxime. Avibactam and OP0595 bound covalently to TLA-3 via the Ser70 residue and made contacts with residues Ser130, Thr235, and Ser237, which are conserved in ESBLs. Additionally, the sulfate group of the inhibitors formed polar contacts with amino acid residues in a positively charged pocket of TLA-3. Our findings provide a structural template for designing improved diazabicyclooctane-based inhibitors that are effective against ESBL-producing Enterobacteriaceae.

Comparison of activities of beta-lactam antibiotics against Streptococcus pneumoniae with recombinant penicillin-binding protein genes from a penicillin-resistant strain.

We investigated the antibacterial activities of 19 beta-lactams against three recombinant bacterial strains, in which three penicillin-binding protein genes, pbp2x, pbp1a, and pbp2b, from penicillin-resistant Streptococcus pneumoniae (PRSP), were transformed to a penicillin-susceptible strain. By the acquisition of the pbp2x gene from PRSP, the minimum inhibitory concentrations (MICs) of third-generation cephalosporins were increased more than eight fold. When the strain acquired the PRSP pbp1a gene in addition to pbp2x, the MICs of all tested beta-lactams increased 2- to 16-fold. When the strain acquired the PRSP pbp2b gene in addition to pbp2x and pbp1a, the MICs of penicillins and carbapenems increased 4- to 16-fold. However, two novel carbapenems, ME1036 and L-036, showed excellent antibacterial activities against these recombinant strains, as well as against the parent PRSP.

Therapeutic effect of ME1036 on endocarditis experimentally induced by methicillin-resistant Staphylococcus aureus.

The efficacy of ME1036, a novel parenteral carbapenem, was compared with that of vancomycin by using a rabbit model of methicillin-resistant Staphylococcus aureus (MRSA) endocarditis. Compared with vancomycin, ME1036 reduced the bacterial counts in the vegetations at a lower dosage or over a shorter period of administration when it was used for the treatment of MRSA endocarditis.

In vitro activities of ME1036 (CP5609), a novel parenteral carbapenem, against methicillin-resistant staphylococci.

ME1036, formerly CP5609, is a novel parenteral carbapenem with a 7-acylated imidazo[5,1-b]thiazole-2-yl group directly attached to the carbapenem moiety of the C-2 position. The present study evaluated the in vitro activities of ME1036 against clinical isolates of gram-positive and gram-negative bacteria. ME1036 displayed broad activity against aerobic gram-positive and gram-negative bacteria. Unlike other marketed beta-lactam antibiotics, ME1036 maintained excellent activity against multiple-drug-resistant gram-positive bacteria, such as methicillin-resistant staphylococci and penicillin-resistant Streptococcus pneumoniae (PRSP). The MICs of this compound at which 90% of isolates were inhibited were 2 microg/ml for methicillin-resistant Staphylococcus aureus (MRSA), 2 microg/ml for methicillin-resistant coagulase-negative staphylococci, and 0.031 microg/ml for PRSP. In time-kill studies with six strains of MRSA, ME1036 at four times the MIC caused a time-dependent decrease in the numbers of viable MRSA cells. The activity of ME1036 against MRSA is related to its high affinity for penicillin-binding protein 2a, for which the 50% inhibitory concentration of ME1036 was approximately 300-fold lower than that of imipenem. In conclusion, ME1036 demonstrated a broad antibacterial spectrum and high levels of activity in vitro against staphylococci, including beta-lactam-resistant strains.

Complete sequences of six penicillin-binding protein genes from 40 Streptococcus pneumoniae clinical isolates collected in Japan.

All six penicillin-binding protein (PBP) genes, namely, pbp1a, pbp1b, pbp2a, pbp2b, pbp2x, and pbp3, of 40 Streptococcus pneumoniae clinical isolates, including penicillin-resistant S. pneumoniae isolates collected in Japan, were completely sequenced. The MICs of penicillin for these strains varied between 0.015 and 8 microg/ml. In PBP 2X, the Thr550Ala mutation close to the KSG motif was observed in only 1 of 40 strains, whereas the Met339Phe mutation in the STMK motif was observed in six strains. These six strains were highly resistant (MICs >/= 2 microg/ml) to cefotaxime. The MICs of cefotaxime for 27 strains bearing the Thr338Ala mutation tended to increase, but the His394Leu mutation next to the SSN motif did not exist in these strains. In PBP 2B, the Thr451Ala/Phe/Ser and Glu481Gly mutations close to the SSN motif were observed in 24 strains, which showed penicillin resistance and intermediate resistance, and the Thr624Gly mutation close to the KTG motif was observed in 2 strains for which the imipenem MIC (0.5 microg/ml) was the highest imipenem MIC detected. In PBP 1A, the Thr371Ser/Ala mutation in the STMK motif was observed in all 13 strains for which the penicillin MICs were >/=1 microg/ml. In PBP 2A, the Thr411Ala mutation in the STIK motif was observed in one strain for which with the cefotaxime MIC (8 microg/ml) was the highest cefotaxime MIC detected. On the other hand, in PBPs 1B and 3, no mutations associated with resistance were observed. The results obtained here support the concept that alterations in PBPs 2B, 2X, and 1A are mainly involved in S. pneumoniae resistance to beta-lactam antibiotics. Our findings also suggest that the Thr411Ala mutation in PBP 2A may be associated with beta-lactam resistance.