Broad-Spectrum Antimicrobial Activity of Oftasecur and Visuprime Ophthalmic Solutions

Abstract

Due to the wide etiology of conjunctivitis, the expensive and time-consuming diagnosis requires new therapeutic strategies with broad-spectrum antimicrobial activity and nonselective mechanisms of action. In this context, eye drops could provide an alternative to conventional antimicrobial therapies. Here, we compare the antibacterial and antiviral activity of Oftasecur and Visuprime, commercially available ophthalmic solutions. Cytotoxicity assay was performed on Vero CCL-81 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Antibacterial efficacy was evaluated on Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae by disk diffusion, broth microdilution methods, and time-killing tests. Furthermore, the antiviral activity against HSV-1 was estimated by co-treatment, cell and viral pretreatment and post-treatment, via plaque reduction assay, fluorescence assessment (GFP-engineered HSV-1), and real-time PCR. After 24 h of exposure, Oftasecur and Visuprime showed a volume-inducing 50% of cytotoxicity of 125 and 15.8 μL, respectively Oftasecur and Visuprime induced 90% antibacterial activity in response to mean volume of 10.0 and 4.4 µL for Gram-positive and Gram-negative strains, respectively. Oftasecur exerted bactericidal action on both bacterial populations, while Visuprime was bacteriostatic on Gram-negative strains and slightly bactericidal on Gram-positive bacteria. A major impact on infectivity occurred by exposure of viral particles to the ophthalmic solutions. In detail, 50% of inhibition was verified by exposing the viral particles to 3.12 and 0.84 μL of Oftasecur and Visuprime, respectively, for 1 h. The reduction of the fluorescence and the expression of the viral genes confirmed the recorded antiviral activity. Due to their high antimicrobial efficiency, Oftasecur and Visuprime could represent a valid empirical strategy for the treatment of conjunctivitis.

Keywords: Gram-negative bacteria; Gram-positive bacteria; HSV-1; Oftasecur; Visuprime; antibacterial activity; antiviral potential; eye drops.

Figure 1

(A) Oftasecur cytotoxicity on Vero CCL-81 cell line after 0.5 (***: p-value = 0.0012, **: p-value = 0.0021, ns: p-value > 0.88), 2 (***: p-value < 0.0010, **: p-value < 0.0018, ns: p-value > 0.76) and 24 h (****: p-value < 0.0001, ***: p-value < 0.0015, **: p-value < 0.0023). (B) Visuprime cytotoxicity on Vero CCL-81 cell line after 0.5 (**: p-value < 0.0025, ns: p-value > 0.99), 2 (****: p-value < 0.0001, ***: p-value < 0.0010, **: p-value < 0.0018, ns: p-value > 0.76), and 24 h (****: p-value < 0.0001, ***: p-value < 0.0015, **: p-value < 0.0023, ns: p-value > 0.99).

Figure 2

Oftasecur (O) and Visuprime (V) inhibition zone against E. coli (A), K. pneumoniae (B), P. aeruginosa (C), S. aureus (D), and S. epidermidis (E). Piperacillin (30 μg) was used as CTRL+.

Figure 3

Antimicrobial activity of Oftasecur against (A) S. aureus (****: p-value < 0.0001, ***: p-value < 0.0014, ns: p-value > 0.71); (B) S. epidermidis (****: p-value < 0.0001, ***: p-value = 0.0009); (C) K. pneumoniae (****: p-value < 0.0001, **: p-value = 0.0012); (D) P. aeruginosa (****: p-value < 0.0001, ***: p-value = 0.0008, **: p-value = 0.0016, *: p-value = 0.023); (E) E. coli (****: p-value < 0.0001, ***: p-value = 0.0006, **: p-value = 0.0019).

Figure 4

Antimicrobial activity of Visuprime against (A) S. aureus (****: p-value < 0.0001, **: p-value = 0.0013); (B) S. epidermidis (****: p-value < 0.0001); (C) K. pneumoniae (****: p-value < 0.0001, ***: p-value = 0.0002); (D) P. aeruginosa (****: p-value < 0.0001); (E) E. coli (****: p-value < 0.0001, ***: p-value = 0.0006).

Figure 5

Oftasecur and Visuprime killing kinetics on (A) E. coli and (B) S. aureus.

Figure 6

Antiviral activity of Oftasecur against HSV-1 in four plaque reduction assays: (A) co-treatment assay (****: p-value < 0.0001, ***: p-value = 0.0004); (B) pre-virus assay (****: p-value < 0.0001); (C) cell pretreatment assay (****: p-value < 0.0001, **: p-value = 0.0063, *: p-value = 0.0208, ns: p-value > 0.1044); (D) post-treatment assay (****: p-value < 0.0001, *: p-value = 0.0187, ns: p-value > 0.0600). (E) Pre-virus assay after 30, 10, and 1 min of exposure (****: p-value < 0.0001, ***: p-value = 0.0002, **: p-value = 0.0073; *: p-value = 0.039).

Figure 7

Antiviral activity of Visuprime against HSV-1 in four plaque reduction assays: (A) co-treatment assay (****: p-value < 0.0001, **: p-value = 0.0065); (B) pre-virus assay (****: p-value < 0.0001; **: p-value = 0.0086); (C) cell pretreatment assay (****: p-value < 0.0001, **: p-value = 0.0086, *: p-value = 0.0109, ns: p-value > 0.06280); (D) post-treatment assay (****: p-value < 0.0001, **: p-value = 0.0075, ns: p-value > 0.1163). (E) Pre-virus assay after 30, 10, and 1 min of exposure (****: p-value < 0.0001, ***: p-value = 0.0003, **: p-value = 0.0074; *: p-value = 0.069).

Figure 8

Antiviral activity of Oftasecur against GFP-engineered HSV-1. Plaques were viewed with bright-field and fluorescent microscopy: treatments at a 12.5 (A) and 3.12 (B) µL; CTRL−: cells infected with the virus (C); CTRL+: cells uninfected (D). Antiviral activity of Visuprime against GFP-engineered HSV-1. Plaques were viewed with bright-field and fluorescent microscopy: treatments at a 6.25 (E) and 1.56 (F) µL; CTRL−: cells infected with the virus (G); CTRL+: cells uninfected (H).

Figure 9

Virus pretreatment with Oftasecur (A) and Visuprime (B) against GFP-engineered HSV-1. The fluorescence signal was read at the excitation wavelength of 395 nm and the emission wavelength of 509 nm. (A): (****: p-value < 0.0001, ***: p-value = 0.0007, **: p-value = 0.0063; ns: p-value = 0.1300); (B): (****: p-value < 0.0001, ***: p-value = 0.0004, *: p-value = 0.0255; ns: p-value = 0.2724).

Figure 10

Quantification of mRNA levels of UL27 and UL54 expressed in HSV-1 infected Vero CCL-81 cells after exposure to Oftasecur (A,B) and Visuprime (C,D) in different stages of viral infection. Data are presented as the ratio of reference genes (GAPDH) to target genes.