Two Newly Isolated Enterobacter-Specific Bacteriophages: Biological Properties and Stability Studies

Abstract

In an era of antibiotic therapy crisis caused by spreading antimicrobial resistance, and when recurrent urinary tract infections constitute a serious social and medical problem, the isolation and complex characterization of phages with a potential therapeutic application represents a promising solution. It is an inevitable, and even a necessary direction in the development of current phage research. In this paper, we present two newly isolated myoviruses that show lytic activity against multidrug-resistant clinical isolates of Enterobacter spp. (E. cloacae, E. hormaechei, and E. kobei), the genomes of which belong to a poorly represented phage group. Both phages were classified as part of the Tevenvirinae subfamily (Entb_43 was recognized as Karamvirus and Entb_45 as Kanagawavirus). Phage lytic spectra ranging from 40 to 60% were obtained. The most effective phage-to-bacteria ratios (MOI = 0.01 and MOI = 0.001) for both the phage amplification and their lytic activity against planktonic bacteria were also estimated. Complete adsorption to host cells were obtained after about 20 min for Entb_43 and 10 min for Entb_45. The phage lysates retained their initial titers even during six months of storage at both -70 °C and 4 °C, whereas storage at 37 °C caused a complete loss in their activity. We showed that phages retained their activity after incubation with solutions of silver and copper nanoparticles, which may indicate possible synergistic antibacterial activity. Moreover, a significant reduction in phage titers was observed after incubation with a disinfectant containing octenidinum dihydrochloridum and phenoxyethanol, as well as with 70% ethanol. The observed maintenance of phage activity during incubation in a urine sample, along with other described properties, may suggest a therapeutic potential of phages at the infection site after intravesical administration.

Keywords: Enterobacter cloacae; Tevenvirinae subfamily; antibiotic resistance; bacteriophages; myoviruses; phage stability; urinary tract infection.

Figure 1

Plaque morphology on agar plates (upper panel) and electron micrographs of phages (lower panel): (A) phage Entb_43 and (B) phage Entb_45. The photographs of the phages’ plaques were taken using a Samsung ST150F camera. Electronograms of the phages were taken using Kodak/Carestream Electron Microscope, at a magnification of (A) 200,000× or (B) 250,000×; bar in each micrograph indicates 100 nm.

Figure 2

Planktonic cell lysis assay for phage Entb_43 and phage Entb_45. Sample measurements were performed in triplicate in two independent experiments. Error bars represent the standard deviation (±SD) of the mean phage titers.

Figure 3

Kinetics of Entb_43 phage adsorption on E. cloacae 30345 and Entb_45 phage on E. cloacae 29796 at MOI = 0.1. Error bars represent the standard deviation (± SD) of the mean phage titers. Lines represent model used for calculation of adsorption constant; dotted line is 95% confidence interval (CI).

Figure 4

Graphical representation of phage Entb_43 (no. 1), aligned to the most similar known genome of Klebsiella phage vB_KaeM KaAlpha (no. 2, acc. no. MN013084). Green—100%, brown-green—between 30% and 100%, and red—0% local similarity. Lack of color shows a gap in alignment. Annotation is shown for reference. Coding sequences (CDS) are yellow, and tRNA coding sequences are pink.

Figure 5

Graphical representation of phage Entb_45 (no. 1), aligned to the most similar known genome of Enterobacter phage ENC9 (no. 2, acc. no. OL355124.1). Green—100%, brown-green—between 30% and 100%, and red—0% local similarity. Lack of color shows a gap in alignment. Annotation is shown for reference. Coding sequences (CDS) are yellow, and tRNA coding sequences are pink.

Figure 6

Shine–Dalgarno motif (ribosome binding site) in sequences of 100 bp upstream of START codon (included) for phage Entb_43, identified by XSTREME software.

Figure 7

Shine–Dalgarno motif (ribosome binding site) in sequences of 100 bp upstream of the START codon (included) for phage Entb_45, identified by XSTREME software.

Figure 8

Phage lysate stability in silver nanoparticles and copper nanoparticles. Samples at the start of the experiments (0 min) were treated as controls for statistical analysis. Error bars represent the standard deviation (±SD) of the mean phage titers. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 9

Phage titers after one-hour incubation in different pH solutions. Samples incubated in a medium with pH = 7.2 were treated as controls. Error bars represent the standard deviation (±SD) of the mean phage titers. ** p < 0.01; *** p < 0.001.

Figure 10

Long-term stability of Entb_43 and Entb_45 phage lysates under various temperature conditions. Error bars represent the standard deviation (±SD) of the mean phage titers.

Figure 10

Long-term stability of Entb_43 and Entb_45 phage lysates under various temperature conditions. Error bars represent the standard deviation (±SD) of the mean phage titers.

Figure 11

Influence of repeated defrosting on titers of phage lysates with or without 20% glycerol. Error bars represent the standard deviation (±SD) of the mean phage titers.

Figure 12

Phage titers after incubation in a urine sample (after 30 min, 1 h, and 24 h of incubation at 37 °C). Samples at the start of the experiments (0 min) were treated as controls. Error bars represent the standard deviation (± SD) of the mean phage titers. *** p < 0.001.