Abstract

Multidrug-resistant hypervirulent Klebsiella pneumoniae caused by the improper use of antibiotics has become a major threat to the dairy industry and public health, posing a major challenge to the treatment of bovine mastitis. The resurgence of phage (and its lysozyme therapy) provides a novel strategy to combat such infections. In this study, bovine Klebsiella pneumoniae phage vB_Kpn_B01 nucleic acid was used as a template to successfully achieve the prokaryotic expression of its endolysin. The results of protein gene annotation and prediction showed that Lysin K had the function of lyase, and was designated Lysin K. After optimizing the production conditions, Lysin K could be produced in a soluble form after induction with 0.25 mmol/L IPTG at 37 °C for 16 h, and the concentration was 100 μg/mL after purification. The stability test confirmed that Lysin K can maintain stable antibacterial activity over a wide temperature and pH range. The minimum inhibitory concentration (MIC) of Lysin K was 0.01 μg/mL and remained stable in milk. The Lysin K showed strong lysis activity in vitro and could effectively lyse three types of multidrug-resistant hypervirulent Klebsiella pneumoniae. In the mouse mastitis model induced by Klebsiella pneumoniae KP18 (108 CFU/mL), the intervention of Lysin K can significantly reduce the bacterial load, alleviate the inflammatory response, and restore the normal functional structure of the target mammary gland. In summary, Lysin K has valuable application prospect in the prevention and treatment of bovine mastitis caused by drug-resistant Klebsiella pneumoniae and the development of phage lysin preparations.

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