Atomic force microscopy analysis of the Acinetobacter baumannii bacteriophage AP22 lytic cycle

Abstract

Background: Acinetobacter baumannii is known for its ability to develop resistance to the major groups of antibiotics, form biofilms, and survive for long periods in hospital environments. The prevalence of infections caused by multidrug-resistant A. baumannii is a significant problem for the modern health care system, and application of lytic bacteriophages for controlling this pathogen may become a solution.

Methodology/principal findings: In this study, using atomic force microscopy (AFM) and microbiological assessment we have investigated A. baumannii bacteriophage AP22, which has been recently described. AFM has revealed the morphology of bacteriophage AP22, adsorbed on the surfaces of mica, graphite and host bacterial cells. Besides, morphological changes of bacteriophage AP22-infected A. baumannii cells were characterized at different stages of the lytic cycle, from phage adsorption to the cell lysis. The phage latent period, estimated from AFM was in good agreement with that obtained by microbiological methods (40 min). Bacteriophage AP22, whose head diameter is 62±1 nm and tail length is 88±9 nm, was shown to disperse A. baumannii aggregates and adsorb to the bacterial surface right from the first minute of their mutual incubation at 37°C.

Conclusions/significance: High rate of bacteriophage AP22 specific adsorption and its ability to disperse bacterial aggregates make this phage very promising for biomedical antimicrobial applications. Complementing microbiological results with AFM data, we demonstrate an effective approach, which allows not only comparing independently obtained characteristics of the lytic cycle but also visualizing the infection process.

Figure 1. Phage AP22 plaques.

Phage AP22 plaques with opaque haloes on the host A. baumannii 1053 strain.

Figure 2. Infection curves of phage AP22.

Representative curves are based on results from three independent assays of a non-infected A. baumannii culture and cultures infected at MOI of 0.5, 5 and 50. Standard deviations are indicated.

Figure 3. AFM images of bacteriophages AP22.

AFM height images (A, D), section analysis (B, E) and height distribution histograms (C, F) of bacteriophages AP22 deposited on the mica (top row) and HOPG (bottom row) surfaces. Sections are built along the dotted lines on the corresponding AFM images. Insets in A and D demonstrate zoomed bacteriophages from the same image. Top inset in A is presented in deflection channel to emphasize holes in the center of the phages.

Figure 4. AFM images of A. baumannii cells.

AFM height (A) and deflection (B) images of A. baumannii cells deposited on the mica surface. (C) Section analysis built along the horizontal line in (A). (D) Zoomed-in region of A. baumannii surface. The inset in (B) demonstrates zoomed-in region around bacterial aggregate (shown by the white square) with the EPS and pili.

Figure 5. AFM images of A. baumannii cells infected for 1–6 minutes.

Left row: deflection AFM images of A. baumannii cells incubated with bacteriophages AP22 for 1 (A), 3(C), 6 (E) minutes. Right row: zoomed-in three dimensional reconstructions of bacterial surfaces depicted on the left (the zoomed regions are indicated by dotted circles in the height images). The inset in (A) demonstrates zoomed-in region, shown by the white square.

Figure 6. AFM images of A. baumannii cells infected for 15–60 minutes.

AFM deflection (A, D) and height (G) images of A. baumannii cells incubated with bacteriophages AP22 for 15 (A), 30 (D), 60 (G) minutes. (B), (E) and (H) represent the section analysis along the dotted lines on (A), (D) and (G) correspondingly. (C), (F) and (I) – zoomed-in three dimensional reconstructions of bacterial surfaces depicted on the left.

Figure 7. The density of adsorption of bacteriophages AP22.

The density of adsorption of bacteriophages AP22 on the surfaces of mica and bacteria for the samples incubated during different time periods.