Predicting the outcome of phage-antibiotic synergy in Galleria mellonella model infected with carbapenem-resistant Acinetobacter baumannii

Abstract

Acinetobacter baumannii is an oppotunistic pathogen that causes serious nosocomial infection in intensive care units. Especially, carbapenem-resistant A. baumannii (CRAB) strains has emerged as a life-threatening pathogen in hospital settings. In this study, we isolated eight A. baumannii phages, which cause lysis of CRAB strains, from sewage sample at a hospital in South Korea. Among the phages, phage Bϕ-R2919 and Bϕ-R1888, which exhibited a broad host range and showed synergy with colistin in double disk potentiation test (DDPT), respectively, were studied. These two phages belonging to the Myoviridae family showed high absorption rate (> 98% within 5 min) and burst size (112-398 PFU/ cell). Both phages showed strong host cell lytic activities at high-dose (MOI = 10), and exhibited broad stability at various temperatures (4-50°C), but low stability at all pH values (pH 4-10). Complete genomes of these phages were sequenced using the Illumina platform. The phages have a double-stranded circular DNA genome with a length of approximately 44 kbp and 37% G+C content. Bioinformatics analysis showed that most of the contents of the phage proteins associated with morphogenesis, replication, and lysis were similar between phage Bϕ-R2919 and Bϕ-R1888 genomes in regards to protein sequence identity, but ORFs organization and direction were different. This study was successful to establish DDPT for the detection of synergy between phage and antibiotics using homemade phage disks on modified Mueller-Hinton agar (MHA) plates. Besides, the in vitro turbidity assays were designed to evaluate interaction between the purified phages and colistin, and to compare the efficacy between monotherapy and combination therapy. Because of inherent one well dilution variation with this in vitro assay and possibility of reproducibility error, the method was difficult to interpret results via observation. However, it provided obvious information of bacterial growth by recording OD600 values over 72 hr of incubation. To investigate isolated phages as monotherapy or in combination with colistin against A. baumannii, their activity was assessed in vivo in Galleria mellonella model of CRAB infection. The survival rate in the larvae model can be explained by results obtained from the in vitro methods. In summary, two A. baumannii phages, Bϕ-R2919 and Bϕ-R1888, infecting clinical CRAB strains were isolated and studied in detail on physiological characterizations and whole genome sequence analysis. To the best of our knowledge, this is the first report using DDPT to determine synergy between phage and antibiotics. Additionally, the preliminary in vitro testing provided proof of the interaction between lytic phages and antibiotics against A. baumannii. Overall, this study demonstrates that in vitro testing exhibit a linear relationship with in vivo activity. Further standardization for the in vitro methods is important to direct combination therapy for antimicrobial-resistant organisms.