Development of a novel phage cocktail and its efficacy in controlling carbapenem-resistant Klebsiella pneumoniae in the food production chain

Klebsiella pneumoniae (K. pneumoniae) is a major hospital-acquired and food-borne pathogen, and it is an important cause of pneumonia, septicaemia, and liver abscesses in humans. In recent years, the isolation rate of K. pneumoniae in the food production chain and animal-derived foods has continued to increase, particularly for drug-resistant strains, posing a significant threat to public health and food safety. In this study, two broad-spectrum K. pneumoniae phages, LAL003 and vB_KpnM-YP11 (YP11 for short), were isolated from Daqing, Heilongjiang Province, China. The phages LAL003 and YP11 are stable under varying conditions, including different pH levels, temperatures, ultraviolet light and ethanol. Phages LAL003 and YP11 lysed 54.55% and 75.76% of the isolates, respectively, and the lysis rate of the phage cocktail composed of LAL003 and YP11 reached 87.88%. This phage cocktail has a complementary host range that includes various capsular serotypes and carbapenem-resistant K. pneumoniae (CRKP). Morphological and genomic analyses revealed that both LAL003 and YP11 belong to the Myoviridae family and Jiaodavirus genus. The genomes of LAL003 and YP11 were composed of linear double-stranded DNA (dsDNA) with lengths of 166,210 bp and 166,607 bp, respectively. Notably, there were no virulence factors, antibiotic resistance genes, or lysogenic genes present in the genomes, and the phage cocktail at high titer did not exhibit any toxicity to Galleria mellonella. The research has found that the phage cocktail has advantages in its effectiveness in controlling CRKP in pork, milk, and lettuce. Furthermore, it possesses potent germicidal capabilities in killing bacteria and removing biofilms of K. pneumoniae from various substrates, such as polypropylene, stainless steel, borosilicate glass, and latex. In summary, these results indicate that, due to the safety and efficacy of phage cocktail, it may control the spread of CRKP from animal-derived foods to humans in the production chain, thereby reducing the risk of cross-contamination of food-contact surface in actual production and mitigating potential health threats to consumers.