These data pointed to an important role for Serine 29 in the activity of nisin and as a consequence, complete saturation mutagenesis was undertaken to determine the impact on nisin activity by substituting serine 29 with all the other available 19 standard amino acids. The strategy proved successful in that three more derivatives, S29A, S29D and S29E displayed enhanced activity against a range of bacterial targets. It is important to note that this improved activity was strain variable, providing further evidence that nisin derivatives can be generated with distinct target specificities. For example, studies with K22T revealed it to be more potent than nisin A against veterinary isolates of S. aureus and S. agalactiae but not Listeria monocytogenes, while N20P is also striking by virtue of the target specific nature of its enhanced activity. Similarly, S29D and S29E displayed improved activity against a distinct number of species, being particularly active against lactococci. In contrast, S29A was more potent than nisin A against all Gram positive and Gram negative bacterial targets. While nisin was first approved for use in 1969, its use is likely to increase in the coming years due to the increased customer demand for minimally processed foods lacking artificial or chemical preservatives. A major concern in food safety is the transmission of pathogenic Enterobacteriaceae due to their major roles in foodborne illness. While nisin is a potent anti-Gram positive inhibitor, its activity against Gram negative bacteria is poor. However, nisin can be used in Benzethonium Chloride combination with other synergistic preservation methods, such as organic acids, low pH, high salt concentrations, chelating agents, modified atmosphere packaging, high hydrostatic pressure and thermal treatments, to enhance anti-Gram negative activity. The superior activity of nisin A S29A compared to nisin 
A against Gram negatives, together with its enhanced activity against all Gram positive targets, suggests that S29A could find applications as a food preservative. Nisin is also used in the veterinary industry and has potential as a clinical antimicrobial. Bovine mastitis is the cause of significant economic loss to dairy operations. More importantly, cattle would not be subject to a zero milk discard and a zero meat withhold as a consequence of 4-(Benzyloxy)phenol treatment. Thus the existence of bioengineered nisin derivatives that consistently exhibit enhanced activity against mastitis associated pathogens such as the S. agalactiae and S. aureus RF122 strains utilized in this study is noteworthy. Furthermore, Escherichia coli can cause inflammation of the mammary gland in dairy cows around parturition and during early lactation with striking local and sometimes severe systemic clinical symptoms. The bacterium invades the udder through the teat canal and may cause several cases of death per year in the most severe cases. The enhanced nature of S29A and S29G against Gram negative species such as E. coli as well as the major mastitis-associated Gram positive species, implies that these derivatives could also reduce the potential for economic loss as a result of their increased potency and broader target range. Indeed, the synergism of nisin A in combination with the polymyxin B nonapeptide reported here would suggest a potential use for this potent combination to control bovine mastitis and, potentially, other veterinary and clinical infections. However, further study is required to establish the mechanistic basis for the enhanced activity of the S29G and S29A derivatives relative to nisin A. In particular, studies will focus on further investigating the importance of the OM with respect to their superior activity against Gram negative bacteria, for which nisin is usually considered ineffective.