Evaluation of the antibacterial activity of tilmicosin-SLN against Streptococcus agalactiae: in vitro and in vivo studies

Abstract

Background: Streptococcus and Staphylococcus are the major contagious organisms causing dairy cow mastitis. Our previous studies have demonstrated that solid lipid nanoparticles (SLNs) can effectively enhance the antimicrobial activity of tilmicosin against Staphylococcus. This study aimed to evaluate the antibacterial efficacy of tilmicosin-loaded SLN (Til-SLN) against Streptococcus agalactiae.

Methods: Til-SLN was prepared using a hot homogenization and ultrasonication method as described previously. Til-SLN was labeled with rhodamine B for nanoparticle tracking. In vitro antibacterial experiments were carried out by broth dilution technique. Pharmacokinetics of the drug and distribution of the nanoparticles in mammary gland were studied after subcutaneous injection in Kunming mice. The therapeutic study was conducted in a mouse mastitis model infected with S. agalactiae.

Results: The results showed that the diameter, polydispersity index, zeta potential, encapsulation efficiency, and loading capacity of the nanoparticles were not significantly affected by fluorescence labeling. Til-SLN showed a sustained and enhanced antibacterial activity in vitro. Til-SLN maintained a sustained drug concentration above 17 µg/g for at least 6 days in the mammary gland, as compared with only 3 days for the same amount of tilmicosin phosphate solution. The mean residence time and elimination half-life (T_1/2) of Til-SLN were much longer than those of tilmicosin phosphate solution. Most of the nanoparticles remained at the injection site and a few were transferred to the mammary glands, indicating that the drug was slowly released at the injection site and then distributed to the mammary glands. SLN significantly enhanced the therapeutic efficacy of tilmicosin as determined by lower colony forming unit counts.

Conclusion: These results demonstrate that SLN could effectively enhance the antibacterial activity of tilmicosin against Streptococcus.

Keywords: Streptococcus agalactiae; mastitis; solid lipid nanoparticles; tilmicosin.

Figure 1

Photographs of tilmicosin-SLN and labeled tilmicosin-SLN (magnification was 10×40). Notes: Left, optical microscopy image of tilmicosin-SLN; middle, optical microscopy image of labeled tilmicosin-SLN; and right, fluorescence microscopy image of labeled tilmicosin-SLN. The fluorescence microscopy image of labeled tilmicosin-SLN was obtained at the same area of normal light. Abbreviation: SLN, solid lipid nanoparticle.

Figure 2

Sustained antibacterial activity of native tilmicosin and tilmicosin-SLN (drug concentration: 0.9 μg/mL). Abbreviations: CFU, colony forming unit; SLN, solid lipid nanoparticle.

Figure 3

Concentrations of tilmicosin in mammary glands after s.c. injection (60 mg/kg). Abbreviations: s.c., subcutaneous; SLN, solid lipid nanoparticle.

Figure 4

Fluorescence microscopy images of mammary gland slices. Notes: Left, treated with labeled tilmicosin-SLN; middle, injection site; and right, treated with rhodamine B solution. Abbreviation: SLN, solid lipid nanoparticle.

Figure 5

The number of Streptococcus agalactiae in mammary glands that were infected with 1×10⁷ CFU/100 µL per mammary gland at 1, 3, and 6 days (mean ± SD, n=5). Abbreviation: CFU, colony forming unit.

Figure 6

CFU counts in mammary glands after treatment. Note: The horizontal line indicates the CFU detection limit. Abbreviations: CFU, colony forming unit; IC, infected control group and treated with 0.9% [w/v] NaCl solution; SLN, solid lipid nanoparticle.