A Bioengineered Nisin Derivative to Control Biofilms of Staphylococcus pseudintermedius

peer-reviewedAntibiotic resistance and the shortage of novel antimicrobials are among the biggest challenges facing society. One of the major factors contributing to resistance is the use of frontline clinical antibiotics in veterinary practice. In order to properly manage dwindling antibiotic resources, we must identify antimicrobials that are specifically targeted to veterinary applications. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many gram-positive bacteria, including human and animal pathogens such as Staphylococcus, Bacillus, Listeria, and Clostridium. Although not currently used in human medicine, nisin is already employed commercially as an anti-mastitis product in the veterinary field. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and also against staphylococci and streptococci associated with bovine mastitis. However, newly emerging pathogens such as methicillin resistant Staphylococcus pseudintermedius (MRSP) pose a significant threat in terms of veterinary health and as a reservoir for antibiotic resistance determinants. In this study we created a nisin derivative with enhanced antimicrobial activity against S. pseudintermedius. In addition, the novel nisin derivative exhibits an enhanced ability to impair biofilm formation and to reduce the density of established biofilms. The activities of this peptide represent a significant improvement over that of the wild-type nisin peptide and merit further investigation with a view to their use to treat S. pseudintermedius infections.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards (10/IN.1/B3027 (http://www.sfi.ie). DF would like to acknowledge receipt of a Society for Applied Microbiology (http://www.sfam.org.uk) Students into Work Grant for FL

Colorimetric readings of biofilms.

<p>Viability of <i>S. pseudintermedius</i> 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 (* = <i>p</i> < 0.05).</p

Inhibition of biofilm formation with nisin A and nisin I4V peptides.

<p>(A) Results of treatment of <i>S. pseudintermedius</i> 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 (** = <i>p</i> < 0.01) and (B) Growth curve analysis of strain <i>S. pseudintermedius</i> DK729 in 1X MIC peptides of nisin A (closed square), I4V (closed diamond) and no peptide (open circle).</p

Specific activity of nisin A and nisin I4V against a range of indicator organisms.

<p>Results from minimum inhibitory concentration assays of purified nisin A and nisin I4V against various Gram-positive targets. Values given are identical results from three independent determinations. Fold Difference represents the improvement of I4V compared to nisin against the relevant indicator.</p><p>Specific activity of nisin A and nisin I4V against a range of indicator organisms.</p

Treatment of biofilms with nisin A and nisin I4V peptides.

<p>(A) <i>S. pseudintermedius</i> DK729 and (B) <i>S. pseudintermedius</i> 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 (* = <i>p</i> < 0.05, ** = <i>p</i> < 0.01, *** = <i>p</i> < 0.001).</p

Growth curve analysis of strains in nisin A and nisin I4V peptides.

<p>(A) <i>S. pseudintermedius</i> DSM21284 in 0.26 mg/L of nisin A (closed square), I4V (closed diamond) and no peptide (open circle), and (B) <i>S. pseudintermedius</i> DK729 in 0.52 mg/L of Nisin A (closed square), I4V (closed diamond) and no peptide (open circle) and <b>(C)</b><i>S. intermedius</i> DSM20373 in 0.52 mg/L of Nisin A (closed square), I4V (closed diamond) and no peptide (open circle).</p

Structure of nisin A and deferred antagonism assays of nisin A and nisin I4V.

<p>(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 <i>S</i>. <i>intermedius</i> DSM 20373, <i>S</i>. <i>pseudintermedius</i> DK729 and <i>S</i>. <i>pseudintermedius</i> DSM21284 by the nisin A producing strain <i>L</i>. <i>lactis</i> NZ9800 pDF05 (pCI372-<i>nis</i>A) and the nisin derivative I4V producing strain <i>L</i>. <i>lactis</i> NZ9800 pDF12 (<i>nis</i>A-I4V).</p

Oligonucleotides utilised in this study.

<p>Emboldened sequences represent degenerate codons (N = A+C+G+T, K = G+T, M = A+C). Underlined sequence corresponds to plasmid (pCI372) DNA.</p><p>Oligonucleotides utilised in this study.</p

Morphology of nisin-treated biofilms examined by microscopy.

<p>(A) Examination of <i>S. pseudintermedius</i> DK729 and (B) <i>S. pseudintermedius</i> DSM21284 biofilms (magnification 1000X) after 24 hour treatment with 16X MIC of nisin A (Wt) and nisin I4V peptides.</p