Paranos Paschalis, Vourli Sophia, Pournaras Spyros, Meletiadis Joseph
Clinical Microbiology Laboratory, Attikon University Hospital, Medical School, National and Kapodistrian University of Athens, Rimini 1, Haidari, 12462 Athens, Greece.
Institute of Biosciences and Applications, National Center for Scientific Research "Demokritos", Patr. Gregoriou E & 27 Neapoleos Str, 15341 Agia Paraskevi, Greece.
Pharmaceuticals (Basel). 2025 Feb 27;18(3):343. doi: 10.3390/ph18030343.
Combination therapy with antibiotics and phages has been suggested to increase the antibacterial activity of both antibiotics and phages. We tested the in vitro activity of five antibiotics belonging to different classes in combination with lytic bacteriophages against multidrug-resistant metallo-β-lactamase (MBL)-producing isolates. A total of 10 non-repetitive well-characterized MBL-producing isolates (5 NDM, 5 VIM) co-resistant to aminoglycosides and quinolones were used. Phage-antibiotic interactions were assessed using an ISO-20776-based broth microdilution checkerboard assay in 96-well microtitration plates. Two-fold dilutions of colistin (8-0.125 mg/L), ciprofloxacin, meropenem, aztreonam, and amikacin (256-4 mg/L) were combined with ten-fold dilutions of five different phages (5 × 10-5 × 10 PFU/mL) belonging to , , and genus. Plates were incubated at 35 ± 2 °C for 24 h, and the minimum inhibitory concentration of antibiotics (MIC) and phages (MIC) were determined as the lowest drug and phage concentration, resulting in <10% growth based on photometric reading at 550 nm. Interactions were assessed based on the fractional inhibitory concentration index (FICi) of three independent replicates and clinical relevance based on the reversal of phenotypic resistance. The statistical significance of each drug alone and in combination with phages was assessed using GraphPad Prism 8.0. Synergistic and additive interactions were found for 60-80% of isolates for all drugs. FICis were statistically significantly lower than 0.5 for colistin ( = 0.005), ciprofloxacin ( = 0.02), meropenem ( = 0.003), and amikacin ( = 0.002). Interactions were found at clinically achievable concentrations for colistin, meropenem, and amikacin, and a reversal of phenotypic resistance was observed for most strains (63-64%) for amikacin and meropenem. Antagonism was found for few isolates with all antibiotics tested. Phage vB_PaerM_AttikonH10 and vB_PaerP_AttikonH4 belonging to and genus, respectively, showed either synergistic (FICi ≤ 0.35) or additive effects with most antibiotics tested. Synergy was observed for most drugs and phages with amikacin, showing strong synergy and reversal of phenotypic resistance against most isolates. Taking into account the wide utility of jumbo phages obtained, the findings of vB_PaerM_AttikonH10 in combination with different classes of antibiotics can enhance the activity of currently ineffective antibiotics against MBL-producing isolates.
抗生素与噬菌体联合治疗被认为可增强抗生素和噬菌体的抗菌活性。我们测试了五类不同抗生素与裂解性噬菌体联合对产多重耐药金属β-内酰胺酶（MBL）菌株的体外活性。共使用了10株特征明确的非重复产MBL菌株（5株产NDM，5株产VIM），这些菌株对氨基糖苷类和喹诺酮类耐药。采用基于ISO-20776的肉汤微量稀释棋盘法在96孔微量滴定板中评估噬菌体-抗生素相互作用。将黏菌素（8 - 0.125 mg/L）、环丙沙星、美罗培南、氨曲南和阿米卡星（256 - 4 mg/L）的两倍稀释液与属于、和属的五种不同噬菌体（5×10 - 5×10 PFU/mL）的十倍稀释液混合。平板在35±2℃孵育24小时，根据550 nm处的光度读数，将抗生素的最低抑菌浓度（MIC）和噬菌体的最低抑菌浓度（MIC）确定为导致生长<10%的最低药物和噬菌体浓度。基于三个独立重复的分数抑菌浓度指数（FICi）评估相互作用，并基于表型耐药性的逆转评估临床相关性。使用GraphPad Prism 8.0评估每种药物单独及与噬菌体联合使用的统计学意义。所有药物60 - 80%的菌株存在协同和相加相互作用。黏菌素（P = 0.005）、环丙沙星（P = 0.02）、美罗培南（P = 0.003）和阿米卡星（P = 0.002）的FICi在统计学上显著低于0.5。在黏菌素、美罗培南和阿米卡星临床可达到的浓度下发现了相互作用，并且在阿米卡星和美罗培南的大多数菌株（63 - 64%）中观察到表型耐药性的逆转。在所测试的所有抗生素中，仅少数菌株存在拮抗作用。分别属于和属的噬菌体vB_PaerM_AttikonH10和vB_PaerP_AttikonH4与大多数测试抗生素表现出协同（FICi≤0.35）或相加作用。大多数药物和噬菌体与阿米卡星表现出协同作用，对大多数菌株表现出强烈的协同作用和表型耐药性的逆转。考虑到所获得的巨型噬菌体的广泛用途，vB_PaerM_AttikonH10与不同类抗生素联合的研究结果可增强目前对产MBL菌株无效的抗生素的活性。
