Fig 1.
Structure of nisin A and deferred antagonism assays of nisin A and nisin I4V.
(A) Residues are represented in the single letter code. Post translational modifications are indicated as follows, Dha: dehydroalanine, Dhb: dehydrobutyrine, Abu: 2-aminobutyric acid, A-A: lanthionine, Abu-A: 3-methyllanthionine. (B) Growth inhibition of S. intermedius DSM 20373, S. pseudintermedius DK729 and S. pseudintermedius DSM21284 by the nisin A producing strain L. lactis NZ9800 pDF05 (pCI372-nisA) and the nisin derivative I4V producing strain L. lactis NZ9800 pDF12 (nisA-I4V).
Table 1.
Oligonucleotides utilised in this study.
Table 2.
Deferred antagonism assays of L. lactis NZ9800 strains producing nisin A (wild type control) and the nisin derivative I4V against representative strains of S. pseudintermedius and S. intermedius.
Table 3.
Specific activity of nisin A and nisin I4V against a range of indicator organisms.
Fig 2.
Growth curve analysis of strains in nisin A and nisin I4V peptides.
(A) S. pseudintermedius DSM21284 in 0.26 mg/L of nisin A (closed square), I4V (closed diamond) and no peptide (open circle), and (B) S. pseudintermedius DK729 in 0.52 mg/L of Nisin A (closed square), I4V (closed diamond) and no peptide (open circle) and (C) S. intermedius DSM20373 in 0.52 mg/L of Nisin A (closed square), I4V (closed diamond) and no peptide (open circle).
Fig 3.
Inhibition of biofilm formation with nisin A and nisin I4V peptides.
(A) Results of treatment of S. pseudintermedius DK729 with 1, 1/2, 1/4, 1/8 and 1/16X MIC of nisin A and nisin I4V peptides for 24 hrs prior to biofilm formation. The amount of biofilm was quantified by measuring the OD595 of crystal violet dissolved in acetic acid. The means and standard deviations of triplicate determinations are presented. Asterisks indicate statistically significant differences (Student’s t-test) between peptides used at similar concentration (** = p < 0.01) and (B) Growth curve analysis of strain S. pseudintermedius DK729 in 1X MIC peptides of nisin A (closed square), I4V (closed diamond) and no peptide (open circle).
Fig 4.
Treatment of biofilms with nisin A and nisin I4V peptides.
(A) S. pseudintermedius DK729 and (B) S. pseudintermedius DSM 21284 with 1, 2, 4, 8 and 16X MIC of nisin A and nisin I4V peptides for 24 hrs as evaluated by crystal violet (CV) staining. The amount of biofilm was quantified by measuring the OD595 of CV dissolved in acetic acid. The means and standard deviations of triplicate determinations are presented. Asterisks indicate statistically significant differences (Student’s t-test) between peptides used at similar concentration (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
Fig 5.
Colorimetric readings of biofilms.
Viability of S. pseudintermedius DK729 following treatment with 16X MIC of nisin A and nisin I4V peptides and untreated control for 24 hrs as evaluated by the XTT (2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assay measured using a microtiter plate reader. Asterisks indicate statistically significant differences (Student’s t-test) between peptides used at similar concentration (* = p < 0.05).
Fig 6.
Morphology of nisin-treated biofilms examined by microscopy.
(A) Examination of S. pseudintermedius DK729 and (B) S. pseudintermedius DSM21284 biofilms (magnification 1000X) after 24 hour treatment with 16X MIC of nisin A (Wt) and nisin I4V peptides.