APTC-C-SA01: A Novel Bacteriophage Cocktail Targeting Staphylococcus aureus and MRSA Biofilms

Abstract

The high infection and mortality rate of methicillin-resistant Staphylococcus aureus (MRSA) necessitates the urgent development of new treatment strategies. Bacteriophages (phages) have several advantages compared to antibiotics for the treatment of multi-drug-resistant bacterial infections, and thus provide a promising alternative to antibiotics. Here, S. aureus phages were isolated from patients and environmental sources. Phages were characterized for stability, morphology and genomic sequence and their bactericidal activity against the biofilm form of methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA was investigated. Four S. aureus phages were isolated and tested against 51 MSSA and MRSA clinical isolates and reference strains. The phages had a broad host range of 82−94% individually and of >98% when combined and could significantly reduce the viability of S. aureus biofilms. The phages had a latent period of ≤20 min and burst size of >11 plaque forming units (PFU)/infected cell. Transmission electron microscopy (TEM) identified phages belonging to the family of Myoviridae. Genomic sequencing indicated the lytic nature of all four phages, with no identified resistance or virulence genes. The 4 phages showed a high complementarity with 49/51 strains (96%) sensitive to at least 2/4 phages tested. Furthermore, the frequency of bacteriophage insensitive mutant (BIM) generation was lower when the phages were combined into the phage cocktail APTC-C-SA01 than for bacteria exposed to each of the phages alone. In conclusion, APTC-C-SA01, containing four lytic S. aureus phages has the potential for further development as a treatment against MSSA and MRSA infections.

Keywords: S. aureus; antimicrobial; bacteriophage; biofilm; phage cocktail.

Figure 1

S. aureus phages’ temperature and pH stability tests. APTC-SA-2, APTC-SA-4, APTC-SA-12, and APTC-SA-13 phage stability testing measured in log₁₀ ^PFU/mL. (A) Phage stability at different temperatures (4, 30, 37, 40, 50, 60, 70, and 80 °C). (B) Phage stability at different pHs (3, 4, 5, 6, 7, 8, 9, 10, 11, and 12). The experiment was repeated 3 times. Bars represent standard deviations (SD). **, p < 0.01; ****, p < 0.0001.

Figure 2

One-step growth curves of S. aureus phages. The log₁₀ ^PFU/mL of phages in the cultures at different time points were tested. Each data point represents the mean from three independent experiments, and the error bars indicate SD. L = the latent period; B = burst size.

Figure 3

Inhibition assays of S. aureus phages. S. aureus ATCC25925 log-phase culture was infected with phages APTC-SA-2 (A), APTC-SA-4 (B), APTC-SA-12 (C), and APTC-SA-13 (D) at MOI = 0 (blue), 0.1 (red), and 1 (green). OD 600 nm was measured at time point = 0 and every 30 min up to time point 390 min. The error bars indicate SD from the results of three independent experiments.

Figure 4

Biofilm assay. S. aureus biofilm (A) ATCC25923, (B) C276 (MSSA), (C) C284 (MSSA), (D) C375(MRSA), and (E) C377(MRSA) were treated with S. aureus phage (10⁸ PFU/mL) for 24 h followed by crystal violet assays to quantify the biofilm biomass. Significance was determined compared to untreated control. Data expressed as mean ± SD for three independent experiments.

Figure 5

Morphology of Staphylococcus phages. Transmission electron microscopy images of S. aureus phage (A) APTC-SA-2, (B) APTC-SA-4, (C)APTC-SA-12, and (D) APTC-SA-13. Scale bar: 100 nm.

Figure 6

S. aureus phages. (A) Map of genomic organization of bacteriophage APTC-SA-2 shown as representative. The open reading frames with predicted annotations are indicated with blue arrows and predicted tRNAs are indicated with green arrows. (B) phytogenic tree of phages. A maximum likelihood phylogenetic tree analysis of APTC-SA-2, APTC-SA-4, APTC-SA-12, APTC-SA-13, and phage K was based on their nucleotide sequences.