Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii

Abstract

Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (10⁷ p.f.u. ml^-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

Keywords: Acinetobacter baumannii; Galleria mellonella; lytic; phage resistance; virulence.

Fig. 1.. (a) Transmission electron microscope images of novel Myovirus’ LemonAid and Tonic, (b) LemonAid plaques on agar in dilution series (p.f.u.=plaque forming units).

Fig. 2.. Alignment of LemonAid lysate reads against the host bacteria, A. baumannii NCTC 13420, showing high coverage of the two prophage Fizzy and Cloudy.

Fig. 3.. (a) Replicate (N=3) in vitro growth curves of A. baumannii NCTC 13240 WT infected with novel bacteriophage, LemonAid (4.6×10⁹) and Tonic (6.6×10⁹), in 1 : 10 serial dilution and the virulence of each phage against A. baumannii, calculated from the growth curves as 1- AUC (phage treatment)/AUC (no phage). (b) In vitro growth of A. baumannii NCTC 13420 and the phage-resistant strains (eight replicates). (c) Comparison of wild-type and phage-resistant A. baumannii variants: growth curve indices: AUC, k (carrying capacity) and r (growth rate). AUC=area under the curve. LB ctrl=LB broth with no bacteria or phage present. Phage ctrl=phage only at highest titre.

Fig. 4.. (a.i) Sketch representation of A. baumannii NCTC 13420 reference genome and the position of two active prophage (Fizzy and Cloudy) and an 8 bp insertion at 2 467 798 bp, (a.ii) an alignment of the wild-type and phage-resistant A. baumannii variants against the reference genome showing the sequence of the region of a gene of unknown function containing the 8 bp insertion and early truncation of the protein when the SNP is present and (a.iii) details of the insertion and its frequency in the different variants in Geneious. (b) Absorption assay: LemonAid p.f.u. ml⁻¹ recorded during 20 min infections against A. baumannii WT (blue) and A. baumannii LemonAid-resistant variant (magenta), to estimate phage adsorption, (c) examples of cell staining and light microscopy, and SEM images of WT A. baumannii and phage-resistant variants.

Fig. 5.. Survival (a) and melanization (b) of G. mellonella larvae inoculated with A. baumannii NCTC 13420 (magenta) and phage-resistant forms of the same strain (LemonAid-resistant – blue; Tonic-resistant – orange).

Fig. 6.. Comparison of (a) survival and (b) melanization across G. mellonella treatment groups: negative controls (1×PBS and phage-only), positive control (inoculation with A. baumannii only) at a dose of 4.6×10⁶ c.f.u., and remedial and prophylactic treatment with LemonAid (at a dose of 4.6×10⁶ p.f.u.) of A. baumannii inoculated larvae. The dotted lines in (a) represent the median survival time of each treatment group. Note, A. baumannii only and remedial treatment groups (N=60), prophylactic phage treatment (N=30).