Characterization of vB_ApiM_fHyAci03, a novel lytic bacteriophage that infects clinical Acinetobacter strains

The genus Acinetobacter includes multiple nosocomial opportunistic pathogens. Within this genus, A. baumannii, A. pittii, and A. nosocomialis are the most frequently isolated species from hospitalized patients around the world [1]. Recently, these bacteria have become a public health concern because of the growing tendency to develop antibiotic resistance [2]. Due to the continued increase in multidrug-resistant (MDR) bacterial strains, there has been a great deal of recent interest in phage therapy research.

fHyAci03 was isolated from a municipal sewage sample collected in Hyvinkää, Finland, using clinical A. pittii strain #5565 (obtained from HUSLAB, Helsinki, Finland) as the host. The morphology of fHyAci03 was examined by transmission electron microscopy as described previously [3] (Supplementary Fig. 1). The dimensions of the prolate head were 111 ± 5.2 nm (length) and 82 ± 5.1 nm (width), and the tail length was 90 ± 4 nm. Dimensions were calculated based on ten virions. Together with the genomic information, the morphological characteristics indicated that fHyAci03 belongs to the family Myoviridae and the subfamily Tevenvirinae. fHyAci03 host range was tested with 48 clinical Acinetobacter strains (Supplementary Table 1). Host range experiments revealed that fHyAci03 could infect two out of three A. nosocomialis strains, and six out of 18 A. pittii strains that were tested. Phage DNA was isolated using an Invisorb Spin Virus DNA Minikit (Stratec Biomedical). Next-generation sequencing was performed at the Institute for Molecular Medicine Finland (FIMM), using a DNA library that was constructed using a Nextera Sample Prep Kit (Illumina, San Diego, CA, USA). Paired-end sequencing was done using an Illumina MiSeq sequencer (Illumina, San Diego, CA, USA) with a read length of 300 nucleotides. Draft sequences were assembled using two pipelines in parallel, Geneious software, version 10.1, and the A5-miseq integrated pipeline [4]. The average whole-genome read coverage was 42.9x. Results from both pipelines were compared to identify a single consensus sequence. To manually verify the fidelity of the assembly, the reads were mapped back to the contigs using Geneious (mean coverage 43.0x, range, 11-96x). The genome was found to be 165,975 bp in length, with a mean GC content of 36.8%. The genome was annotated using Geneious software, RAST [5], BLASTP [6], ARAGORN [7], tRNAscan-SE version 2.0 [8], ResFinder-3.1 [9], and VirulenceFinder-2.0 [10]. fHyAci03 contained 255 predicted genes, eight of which code for tRNAs (Supplementary table S2) and 247 for proteins (Fig. 1). No toxin-, virulence-factor-, antibiotic-resistance-, or lysogeny-related genes were found, indicating that fHyAci03 is strictly lytic and a potential new candidate for phage therapy. A BLASTn search revealed that the most closely related phages belonged to the subfamily Tevenvirinae (Supplementary Table S3). Alignment with EMBOSS Stretcher showed that fHyAci03 is 92.4% identical to A. baumannii phage KARL-1 (MH713599.1) [11]. Prior to the sequence comparisons, the sequence of KARL-1 was rearranged with respect to its orientation and starting point to align maximally with the T-even phages. Genome-wide nucleotide phylogenetic analysis using VICTOR (Virus Classification and Tree Building Online Resource) [12] placed fHyAci03 in the same subcluster with KARL-1 (Fig. 2A). Further phylogenetic analysis based on phage tail fiber amino acid sequences using VICTOR assigned fHyAci03 and KARL-1 to different subclusters (Fig. 2B), reflecting the difference in host species. Comparisons of conserved gene product content between fHyAci03, KARL1, and other members of the subfamily Tevenvirinae, were performed using CoreGenes3.5 [13] (Supplementary Table S4). Based on shared gene content (96.76 % shared homologs) and nucleotide sequence similarity, we suggest that fHyAci03 and KARL-1 could comprise a new genus within the subfamily Tevenvirinae, for which we propose the name “FHyAci03virus” after the first sequenced isolate. To conclude, fHyAci03 is a novel lytic phage that, with further characterization, could represent an interesting new candidate for phage therapy.

The genomic sequence of fHyAci03 has been deposited in the GenBank database under the accession number MH460829.1.