In Vitro and In Vivo Activities of OP0595, a New Diazabicyclooctane, against CTX-M-15-Positive Escherichia coli and KPC-Positive Klebsiella pneumoniae.

Gram-negative bacteria are evolving to produce ß-lactamases of increasing diversity that challenge antimicrobial chemotherapy. OP0595 is a new diazabicyclooctane serine ß-lactamase inhibitor which acts also as an antibiotic and as a ß-lactamase-independent ß-lactam "enhancer" against Enterobacteriaceae Here we determined the optimal concentration of OP0595 in combination with piperacillin, cefepime, and meropenem, in addition to the antibacterial activity of OP0595 alone and in combination with cefepime, in in vitro time-kill studies and an in vivo infection model against five strains of CTX-M-15-positive Escherichia coli and five strains of KPC-positive Klebsiella pneumoniae An OP0595 concentration of 4 µg/ml was found to be sufficient for an effective combination with all three ß-lactam agents. In both in vitro time-kill studies and an in vivo model of infection, cefepime-OP0595 showed stronger efficacy than cefepime alone against all ß-lactamase-positive strains tested, whereas OP0595 alone showed weaker or no efficacy. Taken together, these data indicate that combinational use of OP0595 and a ß-lactam agent is important to exert the antimicrobial functions of OP0595.

OP0595, a new diazabicyclooctane: mode of action as a serine ß-lactamase inhibitor, antibiotic and ß-lactam 'enhancer'.

OBJECTIVES: The production of a growing diversity of ß-lactamases by Gram-negative bacteria challenges antimicrobial chemotherapy. OP0595, discovered separately by each of Meiji Seika Pharma and Fedora Pharmaceuticals, is a new diazabicyclooctane serine ß-lactamase inhibitor that also acts as an antibiotic and as a ß-lactamase-independent ß-lactam 'enhancer'. METHODS: Inhibitory activity against serine ß-lactamases and affinity for PBPs were determined using nitrocefin and Bocillin FL, respectively. MICs alone and in combination with ß-lactam agents were measured according to CLSI recommendations. Morphological changes in Escherichia coli were examined by phase-contrast microscopy. RESULTS: IC50s of OP0595 for class A and C ß-lactamases were <1000 nM, with covalent binding demonstrated to the active-site serine of CTX-M-44 and AmpC enzymes. OP0595 also had direct antibiotic activity against many Enterobacteriaceae, associated with inhibition of PBP2 and conversion of the bacteria into spherical forms. Synergy between OP0595 and ß-lactam agents was seen against strains producing class A and C ß-lactamases vulnerable to inhibition. Lastly, OP0595 lowered the MICs of PBP3-targeted partner ß-lactam agents for a non-ß-lactamase-producing E. coli mutant that was resistant to OP0595 itself, indicating ß-lactamase-independent 'enhancer'-based synergy. CONCLUSIONS: OP0595 acts in three ways: (i) as an inhibitor of class A and C ß-lactamases, covalently binding at their active sites; (ii) as an antibacterial, by inhibiting PBP2 of several Enterobacteriaceae; and (iii) as an 'enhancer' of ß-lactam agents that bind to other PBPs besides PBP2 for several Enterobacteriaceae. OP0595 has considerable potential to overcome resistance when it is combined with various ß-lactam agents.

Identification of novel genes responsible for overexpression of ampC in Pseudomonas aeruginosa PAO1.

The development of resistance to antipseudomonal penicillins and cephalosporins mediated by the chromosomal ampC gene in Pseudomonas aeruginosa is of clinical importance. We isolated piperacillin-resistant mutants derived from P. aeruginosa PAO1 and analyzed two mutants that had an insertion in mpl and nuoN. One mutant, YT1677, was resistant to piperacillin and ceftazidime and had an insertion in mpl, which encodes UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase. The other mutant, YT7988, showed increased MICs of piperacillin, ceftazidime, cefepime, and cefoperazone, and the insertion was mapped to nuoN, which encodes NADH dehydrogenase I chain N. Complementation experiments demonstrated that these mutations resulted in higher levels of resistance to ß-lactams. The expression of genes reported to be involved in ß-lactam resistance was examined by real-time PCR in YT1677 and YT7988 mutants. Overexpression was observed for only ampC, and other genes were expressed normally. Deletion of the ampR gene in YT1677 and YT7988 resulted in decreased expression of ampC, indicating that the mutations in YT1677 and YT7988 affected the expression of ampC through the function of AmpR.

In Vitro Efficacy of Meropenem-Cefmetazole Combination Therapy against New Delhi Metallo-ß-lactamase-producing Enterobacteriaceae.

BACKGROUND: Limited treatment options complicate management of infections with New Delhi metallo-ß-lactamase (NDM)-producing organisms. The efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) was assessed against NDM-producing Enterobacteriaceae. MATERIALS AND METHODS: Twelve Escherichia coli clinical isolates harbouring blaNDM-1 and a positive control E. coli BAA-2469 harbouring blaNDM-1 were studied. Minimum inhibitory concentrations (MICs) of MEM, ertapenem (ERT) and CMZ were determined by broth microdilution. Checkerboard and time-kill assays were performed to confirm the in vitro efficacy of the MEM/CMZ combination. Scanning electron microscopy, kinetic studies and whole-genome sequence analysis were used to determine the antimicrobial resistance mechanisms. RESULTS: MICs of MEM, ERT and CMZ in monotherapy ranged from 8 to 32, 16 to 128, and 32 to 512 µg/mL, respectively. In the checkerboard assay, MEM/ERT resulted in no synergy, whereas MEM/CMZ showed a synergistic effect in all the tested isolates. Furthermore, the MIC of MEM in combination decreased by 2- to 8-fold compared with that of MEM alone. The time-kill study revealed a bactericidal effect in 4 of 13 isolates at 24 h. Scanning electron microscopy showed spheroidisation of the bacterial cell in the MEM/CMZ combination; this was not observed in single antibiotic conditions. Kinetic studies indicated CMZ was a better antagonist for NDM-1 than ERT. Whole-genome sequence analysis did not reveal any explainable differences between isolates susceptible and those non-susceptible to combination therapy. CONCLUSION: In vitro studies showed the potential effectiveness of MEM/CMZ combination therapy against NDM-producing organisms.

Estrogen Receptor-positive Ductal Carcinoma In Situ Frequently Overexpresses HER2 Protein Without Gene Amplification.

Overexpression of human epidermal growth factor receptor 2 (HER2) protein is well known to be more frequent in ductal carcinoma in situ (DCIS) than in invasive ductal carcinoma (IDC). However, the reasons for this difference are poorly understood. On the basis of the high frequency of estrogen receptor-positive (ER+) and HER2-positive (HER2+) DCIS, we hypothesized that this tumor type overexpresses HER2 protein without gene amplification and retrospectively investigated the HER2/neu gene status of 71 ER(+)HER2(+) DCIS, surgically removed during the 2007 to 2017 period, employing fluorescence in situ hybridization (FISH). To compare HER2 protein expressions between in situ and invasive components of individual tumors, 86 pT1mi/1a IDC with predominantly in situ disease were also examined. Furthermore, for comparison of FISH status between in situ and coexisting invasive components, another patient cohort, 78 FISH-positive IDC cases, were employed. To elucidate biological differences among DCIS with various combinations of ER and HER2 protein expressions, we also analyzed public microarray data of mRNA. HER2 gene amplification was observed in 35% of ER(+) and HER2 protein-overexpressing specimens, significantly lower than the 94% in ER-negative (ER-) and HER2 protein-overexpressing specimens (P<0.001). HER2 protein expression was decreased in the invasive component as compared with coexisting in situ portions in 40% of individual tumors, whereas the FISH status of these 2 components was well preserved. Moreover, ER(+) and HER2 protein-overexpressing DCIS showed significantly higher hypoxia-inducible factor-1α protein expression than the ER(+) and HER2 protein-nonoverexpressing tumors (P=0.016). We revealed that ER(+) and HER2 protein-overexpressing DCIS, especially ER-high tumors, frequently overexpress HER2 protein without gene amplification. Our data may provide novel insights for understanding the biology of DCIS.

In vitro and in vivo activities of the diazabicyclooctane OP0595 against AmpC-derepressed Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common cause for healthcare-associated infections, which have been historically treated by antipseudomonal ß-lactam agents in the clinical setting. However, P. aeruginosa has evolved to overcome these ß-lactam agents via multiple endogenous resistance mechanisms, including derepression of the chromosomal cephalosporinase (AmpC). In this article, we investigated the effective concentration of OP0595 for combination with piperacillin, cefepime or meropenem in in vitro susceptibility tests, and the antibacterial activity of cefepime in combination with OP0595 in both in vitro time-kill studies and in vivo murine thigh infection model study with AmpC-derepressed P. aeruginosa. The sufficient combinational concentration of OP0595 was a 4 µg ml-1 with all these three ß-lactam agents. OP0595 increased the antibacterial activity of cefepime in both in vitro and in vivo studies against all strains tested. Taken together, OP0595 is the diazabicyclooctane serine ß-lactamase inhibitor with activity against AmpC-derepressed P. aeruginosa and its combinational use with a ß-lactam agent will provide a new approach for the treatment of P. aeruginosa infections.