Characterization of a Straboviridae phage vB_AbaM-SHI and its inhibition effect on biofilms of Acinetobacter baumannii

Abstract

Acinetobacter baumannii (A. baumannii) is a popular clinical pathogen worldwide. Biofilm-associated antibiotic-resistant A. baumannii infection poses a great threat to human health. Bacteria in biofilms are highly resistant to antibiotics and disinfectants. Furthermore, inhibition or eradication of biofilms in husbandry, the food industry and clinics are almost impossible. Phages can move across the biofilm matrix and promote antibiotic penetration. In the present study, a lytic A. baumannii phage vB_AbaM-SHI, belonging to family Straboviridae, was isolated from sauce chop factory drain outlet in Wuxi, China. The DNA genome consists of 44,180 bp which contain 93 open reading frames, and genes encoding products morphogenesis are located at the end of the genome. The amino acid sequence of vB_AbaM-SHI endolysin is different from those of previously reported A. baumannii phages in NCBI. Phage vB_AbaM-SHI endolysin has two additional β strands due to the replacement of a lysine (K) (in KU510289.1, NC_041857.1, JX976549.1 and MH853786.1) with an arginine (R) (SHI) at position 21 of A. baumannii phage endolysin. Spot test showed that phage vB_AbaM-SHI is able to lyse some antibiotic-resistant bacteria, such as A. baumannii (SL, SL1, and SG strains) and E. coli BL21 strain. Additionally, phage vB_AbaM-SHI independently killed bacteria and inhibited bacterial biofilm formation, and synergistically exerted strong antibacterial effects with antibiotics. This study provided a new perspective into the potential application value of phage vB_AbaM-SHI as an antimicrobial agent.

Keywords: Acinetobacter baumannii; antibiotic; bacterial biofilm; inhibition; phage.

Figure 1

Biological characteristics of phage vB_AbaM-SHI. (A) Bacteriolytic plaques appearing on A. baumannii plates inoculated with phage vB_AbaM-SHI; (B) Phage vB_AbaM-SHI genomic DNA was cleaved by restriction endonucleases EcoRI and Hind III (lanes 4 and 5), but not by NotI and Xhol I (lanes 6 and 7); (C) Morphology of phage vB_AbaM-SHI under transmission electron microscopy (TEM); (D) Growth curve of phage vB_AbaM-SHI propagated in A. baumannii SL. “M”: DNA marker; “SHI”: vB_AbaM-SHI.

Figure 2

Line map of the A. baumannii phage vB_AbaM-SHI genome. In the vB_AbaM-SHI genome track, genes colored in red encode tail and genes colored in green encode head. The arrows represent the ORFs and point in the direction of transcription. Replicative DNA helicase and RNA polymerase were marked with cyan rectangular box, cell lysis was marked with purple rectangular box, baseplate assembly proteins were marked with light green rectangular box, and capsid was marked with yellow rectangular box.

Figure 3

Easyfig output image of the genomic comparison among Acinetobacter phage IME-AB2 (top, NC_041857.1), Acinetobacter phage vB_AbaM_IME285 (middle, MH853786.1) and Acinetobacter phage vB_AbaM-SHI (bottom). Phage genomes are presented by linear visualization with coding regions shown as arrows. ORFs are color-coded according to predicted function: red, tail; orange, structural; green, head; yellow, capsid; blue, terminase; purple, endolysin; grey, hypothetical proteins. The percentage of sequence similarity is indicated by the intensity of the gray color. Vertical blocks between analyzed sequences indicate regions with at least 67% of similarity.

Figure 4

Optimum temperature, pH and MOI for the replication of phage vB_AbaM-SHI. (A) Stability of phage vB_AbaM-SHI at different temperatures; (B) Stability of vB_AbaM-SHI at different pH levels; (C) Optimal multiplicity of infection (MOI) determination.

Figure 5

Scanning electron microscopy (SEM) image of A. baumannii SL colonization in biofilms formed on a round coverslip before and after phage vB_AbaM-SHI and kanamycin sulfate treatment. (A) A. baumannii SL was diluted in the overnight culture (1:250) and cultured for 24 hours before the addition of (B) phage vB_AbaM-SHI (MOI = 0.1) or (C) kanamycin sulfate (10 µg mL^-1); (D) A. baumannii SL was diluted in the overnight culture (1:250) and cultured for 12 hours, and then phage vB_AbaM-SHI (MOI = 0.1) was added and cultured for another 12 hours; (E) A. baumannii SL was diluted in the overnight culture (1:250) and cultured for 12 hours, and then kanamycin sulfate (10 µg mL^-1) was added and cultured for another 12 hours; (F) A. baumannii SL was diluted in the overnight culture (1:250) and cultured for 12 hours, and then both phage vB_AbaM-SHI (MOI = 0.1) and kanamycin sulfate (10 µg mL^-1) were added and cultured for another 12 hours. The colonization of bacteria on coverslip was analyzed using scanning electron microscopy (5, 000× magnification). “A.b”: A. baumannii; “SHI”: vB_AbaM-SHI; “K”: kanamycin sulfate.

Figure 6

Effects of phage vB_AbaM-SHI and kanamycin sulfate on planktonic and bacterial biofilm formation. (A, B) Effects of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) on the growth of A. baumannii SL after pre-cultivation for 12 h (A) and 24 h (B). (C, D) Effects of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) on A. baumannii SL (inoculated at a concentration of 4‰) growth after first being cultured for 12 h, followed by phage A. baumannii SL and kanamycin sulfate (10 µg mL^-1) addition and culturing for 12 h (C) and 24 h (D). (E, F) Effects of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) on A. baumannii SL (inoculated at a concentration of 4‰) biofilm formation after pre-cultivation for 12 h (E) and 24 h (F). (G, H) Effects of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) on A. baumannii SL (inoculated at a concentration of 4‰) biofilm formation after an initial culture for 12 h, followed by the addition of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) and culture for 12 (G) and 24 h (H). *P <0.05, ***P <0.001. “A.b”: A. baumannii; “SHI”: vB_AbaM-SHI; “K”: kanamycin sulfate.

Figure 7

The effect of Phage vB_AbaM-SHI on the biofilm formation of A. baumannii SL. (A-C) Biofilm formed by A. baumannii SL at 24 h, 36 h and 60 h, respectively. (D) Phage vB_AbaM-SHI inhibited the formation of A. baumannii SL biofilm (MOI = 1, 36 h).

Figure 8

Phage vB_AbaM-SHI and kanamycin sulfate inhibit the growth of A. baumannii SL. (A) Effects of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) on the growth of A. baumannii SL (diluted in the overnight culture [1:250]) cultured for 24 h. (B) Effects of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) on A. baumannii SL (diluted in the overnight culture [1:250]) after an initial culture for 12 hours followed by the addition of phage vB_AbaM-SHI and kanamycin sulfate (10 µg mL^-1) and culture for 12 h. ***P <0.001. “A.b”: A. baumannii; “SHI”: vB_AbaM-SHI; “K”: kanamycin sulfate.

Figure 9

Amino acid sequence alignment, phylogenetic analysis and predicted tertiary structure of A. baumannii phage endolysins (A) Amino acid sequence alignment of endolysin proteins among vB_AbaM-SHI, KU510289.1, NC_041857.1, and MH853786.1. (B) Phylogenetic tree of A. baumannii phage endolysins. (The red square denotes the A. baumannii phage vB_AbaM-SHI endolysin). (C) Predicted tertiary structure of the A. baumannii phage endolysin protein. Superposition of vB_AbaM-SHI (green), KU510289.1 (cyano), NC_041857.1 (yellow), and MH853786.1 (purple) structures (https://swissmodel.expasy.org/templates/2nr7.1), which was reproduced/adapted from Structural Genomics, the crystal structure of putative secretion activator protein from Porphyromonas gingivalis W83, by Tan, K., Bigelow, L., Gu, M., Joachimiak, A., Midwest Center for Structural Genomics (MCSG), https://swissmodel.expasy.org/templates/2nr7.1, licensed CC-BY-SA-4.0. “SHI”: vB_AbaM-SHI.