Novel Phage Lysin Abp013 against Acinetobacter baumannii

Abstract

As antimicrobial resistance (AMR) continues to pose an ever-growing global health threat, propelling us into a post-antibiotic era, novel alternative therapeutic agents are urgently required. Lysins are bacteriophage-encoded peptidoglycan hydrolases that display great potential as a novel class of antimicrobials for therapeutics. While lysins against Gram-positive bacteria are highly effective when applied exogenously, it is challenging for lysins to access and cleave the peptidoglycan of Gram-negative bacteria due to their outer membrane. In this study, we identify a novel phage lysin Abp013 against Acinetobacter baumannii. Abp013 exhibited significant lytic activity against multidrug-resistant strains of A. baumannii. Notably, we found that Abp013 was able to tolerate the presence of human serum by up to 10%. Using confocal microscopy and LIVE/DEAD staining, we show that Abp013 can access and kill the bacterial cells residing in the biofilm. These results highlight the intrinsic bacteriolytic property of Abp013, suggesting the promising use of Abp013 as a novel therapeutic agent.

Keywords: Acinetobacter baumannii; endolysin; multidrug resistance; novel antibacterial agent; phage lysin.

Figure 1

Bioinformatic analysis of Abp013. (A) Schematic diagram of the domain organization of Abp013. The glycoside hydrolase family 108 (net charge: +2) is located between amino acid residues 7 and 95, while the peptidoglycan-binding domain (net charge: +3) is located between residues 98 and 161. (B) Bootstrap consensus tree showing the phylogenetic relationship of Abp013 and previously reported Gram-negative lysins. The nucleotide sequences were aligned via ClustalW, and the evolutionary history was inferred via the Neighbor–Joining tree method using MEGA11 software.

Figure 2

Purification of Abp013. (A) SDS-PAGE (12%) analysis of Abp013. Gel shows a highly purified 19.5 kDa Abp013 containing a C-terminal 6x HIS needed for affinity chromatography purification. Characterization of Abp013. The dose–response of Abp013 was determined (B) and the lytic activity of Abp013 was tested at various pH buffers (C) and under different salt concentrations (D). The tolerance of Abp013 towards human serum were tested as well (E). The number of log10 CFU/mL was determined through plating 10-fold serial dilution in a bactericidal assay and compared with buffer-treated negative controls. Statistical significance was determined by a two-tailed Student’s t-test with Welch’s correction. * p < 0.05; ** p < 0.01. The dashed line indicates the limit of detection. The experiments were carried out in biological duplicates; error bars represent the standard deviation.

Figure 3

Lytic spectra of Abp013. A total of 4 A. baumanniii, 1 A. radioresistens, 4 P. aeruginosa, and 4 K. pneumoniae strains were incubated with 100 μg/mL of Abp013 in 20 mM sodium phosphate buffer, pH 6.0, for an hour at 37 °C. The number of residual log10 CFU/mL was determined through plating 10-fold serial dilution in a bactericidal assay and compared with buffer-treated negative controls. Statistical significance was determined by a two-tailed Student’s t-test with Welch’s correction. * p < 0.05; ** p < 0.01. The dashed line indicates the limit of detection. The experiments were carried out in duplicates; error bars represent the standard deviation.

Figure 4

The effect of 3 h treatment of Abp013 on A. baumannii ATCC 17961 biofilm that has been pre-grown for 3 h (A) or 24 h (B). Both planktonic and biofilm cells were collected from the same sample well, with planktonic cells referring to the suspended cells present in the treatment buffer prior to the washing and collection of biofilm cells. Statistical significance was determined using a two-way ANOVA with multiple comparison across treatment groups. * p < 0.05; **** p < 0.0001. The dashed line indicates the limit of detection. A total of 4 and 2 independent experiments were carried out for 3 h (A) and 24 h (B) biofilms, respectively, each with 2 technical replicates. Each data point represents the averaged data of an independent experiment.

Figure 5

Representative confocal image of untreated control (A), buffer-treated control (B), 4 µg/mL of colistin (C), and 1.6 mg/mL Abp013 (D), and the corresponding percentage of live cells (E) based on the analysis of at least 3 images for each treatment sample.