# A novel broad-spectrum phage lysin CP02 for biocontrol of Clostridium perfringens in poultry meat

## TL;DR

This study introduces a new phage lysin, CP02, that effectively kills all tested strains of Clostridium perfringens and shows promise for controlling this foodborne pathogen in poultry meat.

## Contribution

CP02 is the first phage lysin with 100% lytic activity against all toxin types of C. perfringens and demonstrates strong biocontrol potential.

## Key findings

- CP02 lysed 100% of tested C. perfringens strains across all toxin types without affecting non-target bacteria.
- CP02 reduced C. perfringens counts in chicken and duck meat by 2.05–2.59 Lg CFU/mL at 25 °C.
- CP02 showed robust environmental tolerance and inhibited biofilm formation and removal effectively.

## Abstract

Clostridium perfringens (C. perfringens) is a major foodborne pathogen, and the increasing prevalence of antimicrobial-resistant strains necessitates novel control strategies to prevent foodborne illnesses. In this study, ten novel phage lysins were identified in the whole-genome sequences of C. perfringens and C. perfringens phages. The homology of these lysins with other reported C. perfringens phage lysins was no more than 27%. Among these, CP02 exhibited the broadest lytic range, lysing 100% (93/93) of tested C. perfringens strains representing toxin types A, B, C, D, and G in spot assays, while showing no activity against non-target bacteria. Meanwhile, CP02 demonstrated rapid bactericidal activity, achieving a 3.17–5.04 Lg CFU/mL reduction in brain heart infusion (BHI) liquid medium within 15 min at 200 μg/mL. CP02 retained over 80% activity at 4–55 °C and 35.6% activity at 75 °C, maintained over 70% activity across pH 3–10 and NaCl concentrations up to 1000 mM, showing robust environmental tolerance. Furthermore, the activity of CP02 was independent of the presence of metal ions, and D8, Y60, E96 and N135 were identified as essential catalytic sites. CP02 at 50 μg/mL inhibited biofilm formation by 55.88–71.97% and removed 67.28-78.33% of pre-formed mature biofilms. In food matrices, CP02 (200 μg/mL) reduced C. perfringens counts by 2.05–2.59 Lg CFU/mL in chicken breast and duck meat at 25 °C, and by 1.46–2.34 Lg CFU/mL at 4 °C. These results demonstrated that CP02 is a promising biocontrol agent to control C. perfringens contamination in poultry products.

Image 1

•CP02 presents favorable biological properties.•CP02 is the first lysin that can cleave C. perfringens with a 100% lysis rate.•CP02 effectively prevent and eliminate the strong biofilm.•CP02 effectively controls C. perfringens in chicken breast and duck meat.

CP02 presents favorable biological properties.

CP02 is the first lysin that can cleave C. perfringens with a 100% lysis rate.

CP02 effectively prevent and eliminate the strong biofilm.

CP02 effectively controls C. perfringens in chicken breast and duck meat.