Lytic phage of carbapenem-resistant Acinetobacter spp. as an alternative therapeutic agent : purification, whole genome analysis, safety and efficacy testing

Abstract

In recent years, antimicrobial resistance has become a major medical threat worldwide. Among them the rapid increase in carbapenem-resistant Acinetobacter baumannii (CRAB) is one of the particular global issues challenging the health care setting. In order to overcome the problems, bacteriophages are newly reviewed to be an alternative strategy for the control of these pathogens.

In this study, we isolated and purified 15 A. baumannii phages, which cause lysis of CRAB strains, from sewage water at a hospital in South Korea, and six of the A. baumannii phages, phage YMC13/01/C62 ABA BP (phage Bphi-C62), YMC 13/01/R2096 ABA BP (phage Bphi-R2096), YMC11/12/R1215 ABA BP (phage Bphi-R1215), YMC11/12/R2315 ABA BP (phage Bphi-R2315), YMC/09/02/B1251 ABA BP (phage Bphi-B1251) and YMC11/11/R3177 ABA BP (phage Bphi-R3177), were studied.

Characterization of the all phages was conducted by transmission electron microscopy (TEM), host range, adsorption rate, one-step growth curve, temperature/pH stability test and host cell lytic activity test against each host bacteria.

Morphological properties of phages by TEM revealed that the A. baumannii phage Bphi-C62, Bphi-R2096, Bphi-R1215, Bphi-R2315 and Bphi-R3177 belong to the family Myoviridae and only phage Bphi-B1251 belongs to the family Podoviridae.

In the characterization test of the phages, almost all the A. baumannii phages exhibited a broad spectrum against clinical CRAB isolates used in this study, but they did not lyse other bacterial strains including Gram-positive strains. These phages showed high absorption rate (> 95% within 5 min) and bust size (42-286 PFU/cell), and exhibited broad stability at various temperatures (25-70°C) and pH (pH 4-10). All phages showed strong host cell lytic activities at high-dose (MOI = 10).

Complete genomes of all phages were sequenced using the 454-Junior Genome Analyzer. The phages have a double-stranded DNA with a length of approximately 44-47 kbp and 37-39% G+C content, however, genome of phage Bphi-R2096 had 98,170 bp, and lysogenic- or toxin-related genes were not identified in these phage genomes. Bioinformatics analysis showed that the whole genome sequence of phage Bphi-R2096 and Bphi-B1252 had no similarity to other previously reported bacteriophages capable of infecting A. baumannii.

To investigate antibacterial activities of isolated phages against CRAB infection in vivo, we selected two A. baumannii phages, phage Bphi-C62 and Bphi-R2096, which exhibited strong host cell lytic activity in vitro. We evaluated their survival rate, histologic features, bacterial clearance and phage titers, and proinflammatory cytokines in the mouse lung infection model. The phage Bphi-62and Bphi-R2096 improved mouse survival rate and histologic damages of lung, and showed the bacterial clearance in the lung within 3 days after intranasal bacterial challenge. Also, no mortality or serious side effects, such as abnormal physical conditions, signs of toxicity, were observed in the phage-administrated mouse group or high-dose group (1011 PFU/ml) of phage.

In summary, 6 novel A. baumannii phages -phage Bphi-C62, Bphi-R2096, Bphi-R1215, Bphi-R2315, Bphi-B1251 and Bphi-R3177- infecting clinical carbapenem-resistant A. baumannii strains were isolated and studied in detail on physiological characterizations and whole genome sequence analysis. Overall, the results revealed that these phages have a potential as an antimicrobial agent to control the spread of carbapenem-resistant A. baumannii strains in clinical settings. Especially, the in vivo results suggest that the novel A. baumannii phage Bphi-62and phageBphi-R2096 can be considered as a therapeutic agent for infectious disease caused by CRAB strains. To the best our knowledge, this is the first report of the characterization of A. baumannii phages in determining its potential as an alternative therapeutic agent using mouse model of pulmonary infection with CRAB clinical isolates.