微量液体希釈法を用いて臨床分離株に対するアズトレオナム（AZT）、イミペネム（IPM）、レレバクタム（REL）の最小発育濃度（MIC）を測定し薬剤感受性を評価した。IMP産生株において23株中5株はAZT耐性、6株はIPM耐性であった。しかし両者同時に耐性を示す株は存在しなかった。RELは弱い抗菌活性を示した。NDM産生株において6株すべてAZT、IPMに耐性を示した。RELは抗菌活性を示さなかった。
チェッカーボード法を用いてNDM産生K. pneumoniae（ATCC BAA-2473）に対するREL/IPM-AZTの併用効果を評価した。REL/IPMの濃度が64/32 µg/mLの時、AZTのMICは0.016 µg/mLと感性となり相乗効果を示した。様々な濃度比率のREL/IPMとAZTを併用し、Sigmoid Imax modelを用いてREL/IPMとAZTの併用MICの関係式を算出したところ、REL/IPM (1/0.5)-AZT、REL/IPM (1/1.2)-AZTと比較してREL/IPM (1/2)-AZTの際にMIC curveが原点近くに描かれ、RELとIPMの比率は1：2が最適であった。今後、AZT とIMPに対して同時に耐性を示すIMP産生株を見出し同様の検討を行う。
ATCC BAA-2473による好中球大腿部感染マウスモデルにおいて、REL/IPM/AZTを96/192/1200 mg/kg/dayで投与した場合、6回分割で静菌効果、12回分割投与で殺菌効果が得られ、両群間の抗菌効果には有意差があり、時間依存的に抗菌活性を示すことが明らかとなった。
REL/IPM、AZTの濃度測定法を各々LC/MS/MS、HPLCを用いて検討した。検量線においてREL、IPM、AZTはそれぞれ0.02-5、0.01-5 0.5-200 µg/mLの範囲で良好な直線性が得られた。さらに、選択性、キャリーオーバー、真度及び精度に問題がないことが確認された。現在、hollow fiber infection model（HFIM）中において腎機能正常成人患者と同じREL、IPM、AZTの血中濃度推移となるかどうか検証するために、経時的に、HFIM中の薬物濃度を測定している。
Antimicrobial susceptibility was evaluated by measuring the minimum inhibitory concentration (MIC) of aztreonam (AZT), imipenem (IPM), and relebactam (REL) against clinical isolates using a broth microdilution. Five of 23 IMP-producing isolates were resistant to AZT and 6 were resistant to IPM, but none were resistant to both at the same time. REL showed weak antibacterial activities. Six of the NDM-producing strains were resistant to AZT and IPM.
The combination effect of REL/IPM-AZT on NDM-producing K. pneumoniae (ATCC BAA-2473) was evaluated using the checkerboard method. When the concentration of REL/IPM was 64/32 µg/mL, the MIC of AZT was 0.016 µg/mL, which was sensitive, indicating synergistic effects. The equation for the combined MIC of REL/IPM and AZT was calculated using the Sigmoid Imax model for various concentration ratios of REL/IPM and AZT. The MIC curve was drawn closer to the origin for REL/IPM (1/2)-AZT than for REL/IPM (1/0.5)-AZT or REL/IPM (1/1.2)-AZT, and the optimal ratio of REL to IPM was 1:2. In the future, we will find IMP-producing strains that show resistance to AZT and IMP at the same time and conduct a similar study.
In neutropenic murine thigh infection models by ATCC BAA-2473, REL/IPM/AZT administered at 96/192/1200 mg/kg/day showed bacteriostatic effect after 6 divided doses and bactericidal effect after 12 divided doses, with a significant difference in antibacterial activities between the two groups. These antimicrobials were found to have time-dependent antimicrobial activities.
The concentration assays for REL/IPM and AZT were investigated using LC/MS/MS and HPLC, respectively. The calibration curves for REL, IPM, and AZT showed good linearity in the range of 0.02-5, 0.01-5, and 0.5-200 µg/mL, respectively. Furthermore, no problems were observed in selectivity, carryover, precision and accuracy. We are currently measuring the concentrations of REL, IPM, and AZT in the hollow fiber infection model to verify whether the blood concentration profiles are similar to those in adult patients with normal renal function.