Bio-Engineered Nisin with Increased Anti-Staphylococcus and Selectively Reduced Anti-Lactococcus Activity for Treatment of Bovine Mastitis.

Bovine mastitis is a significant economic burden for dairy enterprises, responsible for premature culling, prophylactic and therapeutic antibiotic use, reduced milk production and the withholding (and thus wastage) of milk. There is a desire to identify novel antimicrobials that are expressly directed to veterinary applications, do not require a lengthy milk withholding period and that will not have a negative impact on the growth of lactic acid bacteria involved in downstream dairy fermentations. Nisin is the prototypical lantibiotic, a family of highly modified antimicrobial peptides that exhibit potent antimicrobial activity against many Gram-positive microbes, including human and animal pathogens including species of Staphylococcus and Streptococcus. Although not yet utilized in the area of human medicine, nisin is currently applied as the active agent in products designed to prevent bovine mastitis. Over the last decade, we have harnessed bioengineering strategies to boost the specific activity and target spectrum of nisin against several problematic microorganisms. Here, we screen a large bank of engineered nisin derivatives to identify novel derivatives that exhibit improved specific activity against a selection of staphylococci, including mastitis-associated strains, but have unchanged or reduced activity against dairy lactococci. Three such peptides were identified; nisin A M17Q, nisin A T2L and nisin A HTK.

Structure-activity relationship of synthetic variants of the milk-derived antimicrobial peptide αs2-casein f(183-207).

Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183-207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183-207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193-203) and αs2-casein f(197-207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193-207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.

High-pressure processing--effects on microbial food safety and food quality.

High-pressure processing (HPP) is a nonthermal process capable of inactivating and eliminating pathogenic and food spoilage microorganisms. This novel technology has enormous potential in the food industry, controlling food spoilage, improving food safety and extending product shelf life while retaining the characteristics of fresh, preservative-free, minimally processed foods. As with other food processing methods, such as thermal processing, HPP has somewhat limited applications as it cannot be universally applied to all food types, such as some dairy and animal products and shelf-stable low-acid foods. Herein, we discuss the effects of high-pressure processing on microbial food safety and, to a lesser degree, food quality.

Investigation of the Antimicrobial Activity of Bacillus licheniformis Strains Isolated from Retail Powdered Infant Milk Formulae.

This study investigated the potential antimicrobial activity of ten Bacillus licheniformis strains isolated from retail infant milk formulae against a range of indicator (Lactococcus lactis, Lactobacillus bulgaricus and Listeria innocua) and clinically relevant (Listeria monocytogenes, Staphylococcus aureus, Streptococcus agalactiae, Salmonella Typhimurium and Escherichia coli) microorganisms. Deferred antagonism assays confirmed that all B. licheniformis isolates show antimicrobial activity against the Gram-positive target organisms. PCR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analyses indicated that four of the B. licheniformis isolates produce the bacteriocin lichenicidin. The remaining six isolates demonstrated a higher antimicrobial potency than lichenicidin-producing strains. Further analyses identified a peptide of ~1,422 Da as the most likely bioactive responsible for the antibacterial activity of these six isolates. N-terminal sequencing of the ~1,422 Da peptide from one strain identified it as ILPEITXIFHD. This peptide shows a high homology to the non-ribosomal peptides bacitracin and subpeptin, known to be produced by Bacillus spp. Subsequent PCR analyses demonstrated that the six B. licheniformis isolates may harbor the genetic machinery needed for the synthesis of a non-ribosomal peptide synthetase similar to those involved in production of subpeptin and bacitracin, which suggests that the ~1,422 Da peptide might be a variant of subpeptin and bacitracin.

Identification and characterization of an essential gene in Listeria monocytogenes using an inducible gene expression system.

The Listeria monocytogenes gene lmo1594 is a homolog of the Bacillus subtilis cell division gene ezrA. EzrA is a negative regulator of FtsZ ring formation, which is required for efficient cell division as it regulates the frequency and position of Z-rings in the cell and prevents aberrant polar cell division. Previously identified as a putative high pressure (HP) resistance mechanism; conferring enhanced barotolerance when heterologously expressed against an Escherichia coli background; the aim of the current study was to investigate whether lmo1594 plays a role in listerial barotolerance. When the creation of a deletion mutant proved unsuccessful, the role of lmo1594 was addressed by creating a conditional knockout mutant which demonstrated that the gene is in fact essential for cell survival and growth in L. monocytogenes. In order to investigate the effect of lmo1594 on barotolerance, the gene was over-expressed. The over-expression of lmo1594 increased survival levels in L. monocytogenes treated at 300 MPa, but survival levels similar to those of the wild-type strain were observed when treated at a higher pressure (≥400 MPa). In conclusion, this study reveals for the first time that lmo1594 is absolutely essential for listerial cell growth and survival, and also plays an important role in listerial barotolerance.