Bioengineered Nisin A Derivatives Display Enhanced Activity against Clinical Neonatal Pathogens

Abstract

Neonatal infection is a significant cause of mortality and morbidity in infants. The global incidence of multi-drug resistance continues to rise among neonatal pathogens, indicating a need for alternative treatment strategies. Nisin is an antimicrobial peptide that exhibits broad-spectrum activity against a wide variety of clinical pathogens and can be used in combination with antibiotics to improve their effectiveness. This study examined the activity of nisin and bioengineered derivatives against multi-drug resistant Streptococcus agalactiae and Staphylococcus capitis isolates and investigated the potential synergy between nisin peptides and selected antibiotics. Whole genome sequence analysis of the strains revealed the presence of multi-drug resistant determinants, e.g., macrolide, tetracycline, β-lactam, aminoglycoside, while the S. agalactiae strains all possessed both nsr and nsrFP genes and the S. capitis strains were found to encode the nsr gene alone. Deferred antagonism assays demonstrated that nisin PV had improved antimicrobial activity against all strains tested (n = 10). The enhanced specific activity of this peptide was confirmed using minimum inhibitory concentrations (MIC) (0-4-fold lower MIC for nisin PV) and broth-based survival assays. Combinations of nisin peptides with antibiotics were assessed for enhanced antimicrobial activity using growth and time-kill assays and revealed a more effective nisin PV/ampicillin combination against one S. capitis strain while a nisin A/erythromycin combination displayed a synergistic effect against one S. agalactiae strain. The findings of this study suggest that nisin derivatives alone and in combination with antibiotics have potential as alternative antimicrobial strategies to target neonatal pathogens.

Keywords: Staphylococcus capitis; Streptococcus agalactiae; antibacterial peptide; bioengineered peptide; neonatal infections; nisin.

Figure 1

Schematic structures of the 34 amino acid (AA) pentacyclic peptide nisin A and nisin derivatives used in this study, highlighting the position of the five lanthionine rings (A, B, C, D, E) and the hinge region. Residues are represented in the single letter code and post-translational modified AA are indicated in green as follows: Dha: dehydroalanine, Dhb: dehydrobutyrine and Abu: 2-aminobutyric acid. Nisin PV is indicated in blue. Substitute amino acids are indicated in orange.

Figure 2

Deferred antagonism assay illustrating the zones of inhibition produced by the wild-type nisin A (WT), nisin PV and its derivatives nisin T2A-PV, nisin K12A-PV and nisin M17A-PV against (A) L. lactis MG1614 (B) L. lactis MG1614 pNP40 (C) S. agalactiae CIT 67 (D) S. capitis AV80. All plates were supplemented with sub-inhibitory concentration (10 µg/L) of nisin in the form of nisaplin to ensure induction of the nisin mutants.

Figure 3

RP-HPLC profiles of (A) T2A-PV, (B) K12A-PV and (C) M17A-PV. Purified powder from RP-HPLC fractions were subjected to MALDI-ToF mass spectrometric (MS) analysis (inset) to confirm the expected mass of T2A-PV (3338 Da), K12A-PV (3294 Da) and M17A-PV (3291 Da).

Figure 4

Impact of combinations of nisin derivatives and antibiotics on growth of representative Streptococcus agalactiae isolates. Concentrations used were established from MIC evaluation. Growth curve analysis of (A) S. agalactiae CIT 67 in untreated (black square), 1 µg/mL of nisin A (red circle), 1 µg/mL of nisin PV (blue circle), 1 µg/mL of K12A-PV (orange diamond), 0.33 µg/mL of erythromycin (green triangle), and the combinations of nisin A and erythromycin (pink circle), PV and erythromycin (light blue square), K12A-PV and erythromycin (yellow diamond), (B) S. agalactiae CIT 85 in untreated (black square), 1.5 µg/mL of nisin A (red circle), 1.5 µg/mL of nisin PV (blue circle), 1.5 µg/mL of K12A-PV (orange diamond), 0.024 µg/mL of ampicillin (green triangle), and the combinations of nisin A and ampicillin (pink circle), PV and ampicillin (light blue square), K12A-PV and ampicillin (yellow diamond) and (C) S. agalactiae CIT 87 in untreated (black square), 0.26 µg/mL of nisin A (red circle), 0.26 µg/mL of nisin PV (blue circle), 0.26 µg/mL of K12A-PV (orange diamond), 8.3 µg/mL of gentamicin (green triangle), and the combinations of nisin A and gentamicin (pink circle), PV and gentamicin (light blue square), K12A-PV and gentamicin (yellow diamond). The means and standard deviations of the three independent determinations are presented.

Figure 5

Impact of combinations of nisin derivatives and antibiotics on growth of representative Staphylococcus capitis isolates. Concentrations used were established from MIC evaluation. Growth curve analysis of (A) S. capitis AR18 in untreated (black square), 6.25 µg/mL of nisin A (red circle), 6.25 µg/mL of nisin PV (blue circle), 6.25 µg/mL of K12A-PV (orange diamond), 6.25 µg/mL of penicillin (green triangle), and the combinations of nisin A and penicillin (yellow diamond), PV and penicillin (light blue square), K12A-PV and penicillin (yellow diamond), (B) S. capitis AV80 in untreated (black square), 12.5 µg/mL of nisin A (red circle), 12.5 µg/mL of nisin PV (blue circle), 12.5 µg/mL of K12A-PV (orange diamond), 6.25 µg/mL of ampicillin (green triangle), and the combinations of nisin A and ampicillin (pink circle), PV and ampicillin (light blue square), K12A-PV and ampicillin (outlined square) and (C) S. capitis BA06 in untreated (black square), 3.1 µg/mL of nisin A (red circle), 3.1 µg/mL of nisin PV (blue circle), 3.1 µg/mL of K12A-PV (orange diamond), 6.25 µg/mL of ampicillin (green triangle), and the combinations of nisin A and ampicillin (pink circle), PV and ampicillin (light blue square), K12A-PV and ampicillin (yellow diamond). The means and standard deviations of the three independent determinations are presented.

Figure 6

Time-Kill curve analysis of (A) S. agalactiae CIT 67 S. capitis in untreated (black square), 1.5 µg/mL of nisin A (WT) (red circle), 1.5 µg/mL of nisin PV (blue circle), 0.5 µg/mL of erythromycin (green triangle), and combinations of nisin A and erythromycin (pink circle), and nisin PV and erythromycin (light blue square) and (B) S. capitis AV80 in untreated (black square), 12.5 µg/mL of nisin A (red circle), 12.5 µg/mL of nisin PV (blue square), 6.25 µg/mL of ampicillin (green triangle), and the combinations of nisin A and ampicillin (pink circle), PV and ampicillin (light blue square). The means and standard deviations of three independent determinations are presented.