Characterizing the Biology of Lytic Bacteriophage vB_EaeM_φEap-3 Infecting Multidrug-Resistant Enterobacter aerogenes

Abstract

Carbapenem-resistant Enterobacter aerogenes strains are a major clinical problem because of the lack of effective alternative antibiotics. However, viruses that lyze bacteria, called bacteriophages, have potential therapeutic applications in the control of antibiotic-resistant bacteria. In the present study, a lytic bacteriophage specific for E. aerogenes isolates, designated vB_EaeM_φEap-3, was characterized. Based on transmission electron microscopy analysis, phage vB_EaeM_φEap-3 was classified as a member of the family Myoviridae (order, Caudovirales). Host range determination revealed that vB_EaeM_φEap-3 lyzed 18 of the 28 E. aerogenes strains tested, while a one-step growth curve showed a short latent period and a moderate burst size. The stability of vB_EaeM_φEap-3 at various temperatures and pH levels was also examined. Genomic sequencing and bioinformatics analysis revealed that vB_EaeM_φEap-3 has a 175,814-bp double-stranded DNA genome that does not contain any genes considered undesirable for the development of therapeutics (e.g., antibiotic resistance genes, toxin-encoding genes, integrase). The phage genome contained 278 putative protein-coding genes and one tRNA gene, tRNA-Met (AUG). Phylogenetic analysis based on large terminase subunit and major capsid protein sequences suggested that vB_EaeM_φEap-3 belongs to novel genus "Kp15 virus" within the T4-like virus subfamily. Based on host range, genomic, and physiological parameters, we propose that phage vB_EaeM_φEap-3 is a suitable candidate for phage therapy applications.

Keywords: E. aerogenes; Myoviridae; bacteriophage; genome sequencing; vB_EaeM_φEap-3.

FIGURE 1

Plaques of phage vB_EaeM_φEap-3 on Enterobacter aerogenes strain 3-SP.

FIGURE 2

Morphology of phage vB_EaeM_φEap-3. Transmission electron micrograph of negatively stained phage vB_EaeM_φEap-3 at × 150,000 magnification. The bar indicates 100 nm.

FIGURE 3

One-step growth curve of phage vB_EaeM_φEap-3. Phage vB_EaeM_φEap-3 was grown in an exponential phase culture of Enterobacter aerogenes strain 3-SP. Data points indicate the PFU/mL at different time points. Each data point represents the mean of three independent experiments.

FIGURE 4

pH stability of phage vB_EaeM_φEap-3. Each data point represents the mean of three independent experiments. Standard deviations are shown as vertical lines. Results are expressed as PFU/mL.

FIGURE 5

Thermal stability of phage vB_EaeM_φEap-3. Each data point represents the mean of three independent experiments. Results were expressed as PFU/mL.

FIGURE 6

Multiple alignment of the chromosomes of phage vB_EaeM_φEap-3, coliphages RB16 and RB43, Klebsiella phages KP15 and KP27 obtained using Mauve software http://asap.ahabs.wisc.edu/mauve/. The height of the similarity profile corresponds to the average level of conservation in that region of the genome sequence. Completely white regions represent fragments that are not aligned or contain a particular genome-specific sequence element.

FIGURE 7

Genome map of phage vB_EaeM_φEap-3. The genome map was generated using the CLC Main Workbench, version 6.1.1 (CLC bio, Aarhus, Denmark). Predicted open reading frames are indicated by arrows, with the direction of the arrows representing the direction of transcription.

FIGURE 8

Phylogenetic trees generated based on the (A) large terminase subunit and (B) major capsid proteins of vB_EaeM_φEap-3 and homolog proteins from other members of the Tevenvirinae subfamily. Amino acid sequences were compared using ClustalW, and phylogenetic trees were generated using the neighbor-joining method.