In vitro synergistic activities of cefazolin and nisin A against mastitis pathogens.

First-generation cephalosporins such as cefazolin (CEZ) have been widely used for mastitis treatment in dairy cattle. However, the use of antibiotics results in the presence of antibiotic residues in milk, which is used for human consumption. Nisin A, a bacteriocin produced by Lactococcus lactis, has been used as a broad-spectrum food preservative for over 50 years. Therefore, a combination of CEZ and nisin A might provide an extended activity spectrum against mastitis pathogens and reduce the antibiotic dose for mastitis treatment. This study aimed to evaluate the combined effect of CEZ and nisin A against mastitis pathogens using the checkerboard and time-kill assays. In the checkerboard assay, the CEZ-nisin A combination exhibited a synergistic effect against Staphylococcus aureus (n=20/20) and Enterococcus faecalis (n=13/18), and meanwhile exhibited a mostly additive effect against Staphylococcus intermedius (n=12/20), Streptococcus agalactiae (n=10/10), Streptococcus dysgalactiae (n=18/18), and Escherichia coli (n=14/18). There were no indifferent or antagonistic effects between CEZ and nisin A. In the time-kill assay, the CEZ-nisin A combination at 0.5 × or 1 × minimum inhibitory concentration exhibited synergistic reduction of bacterial growth by over 3 log10 colony forming units per ml relative to that observed with either antimicrobial substance alone. These results suggest that the CEZ-nisin A combination can be used for developing an intramammary infusion for mastitis treatment, with lower antibiotic concentrations than normal.

Cationic Lipid Content in Liposome-Encapsulated Nisin Improves Sustainable Bactericidal Activity against Streptococcus mutans.

An oral infectious disease, dental caries, is caused by the cariogenic streptococci Streptococcus mutans. The expected preventive efficiency for prophylactics against dental caries is not yet completely observed. Nisin, a bacteriocin, has been demonstrated to be microbicidal against S. mutans, and liposome-encapsulated nisin improves preventive features that may be exploited for human oral health. Here we examined the bactericidal effect of charged lipids on nisin-loaded liposomes against S. mutans and inhibitory efficiency for insoluble glucan synthesis by the streptococci for prevention of dental caries. Cationic liposome, nisin-loaded dipalmitoylphosphatidylcholine/phytosphingosine, exhibited higher bactericidal activities than those of electroneutral liposome and anionic liposome. Bactericidal efficiency of the cationic liposome revealed that the vesicles exhibited sustained inhibition of glucan synthesis and the lowest rate of release of nisin from the vesicles. The optimizing ability of cationic liposome-encapsulated nisin that exploit the sustained preventive features of an anti-streptococcal strategy may improve prevention of dental caries.

Sustainable inhibition efficacy of liposome-encapsulated nisin on insoluble glucan-biofilm synthesis by Streptococcus mutans.

CONTEXT: Dental caries are an infectious oral bacterial disease caused by cariogenic streptococci. These streptococci inhabit dental biofilms which comprise insoluble glucans. OBJECTIVE: To prevent dental caries, nisin, a suitable agent active against Gram-positive bacteria, was examined in vitro for its ability to suppress insoluble glucan-biofilm synthesis by cariogenic streptococci. MATERIALS AND METHODS: To investigate glucan-biofilm synthesis by a typical cariogenic streptococcus, Streptococcus mutans 10449, the naked form of nisin was loaded onto a 96-well microplate in vitro model. To prolong the efficacy of nisin as a preventive agent, liposome-encapsulated nisin (nisin-liposome) was examined for its ability to inhibit the synthesis of glucan-biofilms on microplates. RESULTS: Naked nisin (100 pmol) completely suppressed insoluble glucan-biofilm synthesis by S. mutans 10449 following 1 h cultivation in 96-well microplates. The concentration of nisin-liposome required for the efficacious inhibition of glucan-biofilm synthesis was four times lower than that of naked nisin following 2 h cultivation. In particular, nisin-liposome (30 pmol nisin equivalent) prolonged the inhibitory activity of nisin against glucan-biofilm synthesis by S. mutans 10449 for up to 6 h, while naked nisin (30 pmol) gradually lost this inhibitory activity over the same period. In vitro release assay of nisin from the liposome showed that 76% nisin was released within 6 h. DISCUSSION AND CONCLUSION: The findings indicate the usefulness of nisin-liposome for the sustained release of nisin. Thus, nisin-liposome could play a potential role in preventive medicine as an inhibitor of the glucan-biofilm synthesis.

Dual antibacterial mechanisms of nisin Z against Gram-positive and Gram-negative bacteria.

Nisin, an amphipathic antibiotic peptide, is produced by a number of strains of Lactococcus lactis subsp. lactis. It has been employed as a food preservative as it has a high antibacterial activity with a relatively low toxicity for humans. Nisin is known to exert a high antibacterial activity against Gram-positive but not Gram-negative bacteria. However, purified nisin Z was found to show an antibacterial activity both against Gram-positive and Gram-negative bacteria. To clarify the mechanisms of activity, nisin Z and purified nisin Z were tested for their antibacterial activities in a high-salt environment. The activity of nisin Z against Staphylococcus aureus was stable even in the presence of NaCl at 100 mM, showing ca. 2log colony-forming unit (CFU) reduction. In contrast, the activity of nisin Z against Escherichia coli was highly sensitive to the same concentration of NaCl, and CFU reduction was not observed. Furthermore, purified nisin Z caused the permeabilisation both of S. aureus and E. coli cytoplasmic membranes. The permeabilisation of E. coli but not S. aureus cytoplasmic membranes was remarkably reduced in a high-salt environment. Moreover, vancomycin inhibited the nisin Z-induced permeabilisation of the S. aureus cytoplasmic membrane. These results suggest that nisin Z utilises two distinct mechanisms of antibacterial activity: a high-salt-sensitive mechanism for E. coli and a high-salt-insensitive mechanism for S. aureus.