To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus. Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure. IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

Bacillus cereus is a spore-forming, Gram-positive bacterium and is a major food-borne pathogen. A B. cereus-specific bacteriophage PBC4 was isolated from the soil of a stock farm, and its genome was analyzed. PBC4 belongs to the Siphoviridae family and has a genome consisting of 80 647-bp-long double-stranded DNA, including 123 genes and two tRNAs. LysPBC4, the endolysin of PBC4, has an enzymatically active domain (EAD) on its N-terminal region and a putative cell wall-binding domain (CBD) on its C-terminal region, respectively. Although the phage PBC4 showed a very limited host range, LysPBC4 could lyse all of the B. cereus strains tested. However, LysPBC4 did not kill other bacteria such as B. subtilis or Listeria, indicating that the endolysin has specific lytic activity against the B. cereus group species. Furthermore, LysPBC4_CBD fused with enhanced green fluorescent protein (EGFP) could decorate limited strains of B. cereus group, suggesting that the LysPBC4_CBD may be a promising material for specific detection of B. cereus.

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Since the first food-borne outbreak of Salmonella enterica serovar Bareilly in the UK (2010), it has been recognized as a new type of food-borne pathogen in S. enterica. To detect and characterize this new serovar pathogen in South Korea, a total of 175 Salmonella strains was isolated and 31 isolates were identified as S. Bareilly from various food-borne outbreaks between 2014 and 2018. While pulsed-field gel electrophoresis (PFGE) analysis using XbaI revealed two major groups (A and B) each with two subgroups (A1, A2/B1, B2), average nucleotide identity (ANI), single nucleotide polymorphism (SNP), and in silico multilocus sequence typing (MLST) analyses confirmed only two major groups. Interestingly, extended SNP analysis with 67 S. Bareilly strains from outbreaks in other countries revealed that A group strains between 2014 and 2016 shared a close evolutionary relationship with the strains from outside of South Korea; however, the B group strains in 2018 were located in a separate SNP tree branch. These findings suggest that the A group may share common ancestor with the strains of previous outbreaks in the UK or other countries, while the B group is a new genotype. Comparative virulence factor (VF) analysis between the A and B group strains showed that S. Bareilly in the B group has more various than that of the A group. A comparative biofilm formation assay supports for this, which B group strain GG-21 has higher biofilm formation activity than A group strain GG-07. Antibiotic susceptibility test of 31 S. Bareilly strains revealed high susceptibility to 17 tested antibiotics, suggesting that S. Bareilly can be easily treated by antibiotics.