Bacteriophage PBC1 and Its Endolysin as an Antimicrobial Agent against Bacillus cereus

Abstract

Bacillus cereus is an opportunistic human pathogen responsible for food poisoning and other, nongastrointestinal infections. Due to the emergence of multidrug-resistant B. cereus strains, the demand for alternative therapeutic options is increasing. To address these problems, we isolated and characterized a Siphoviridae virulent phage, PBC1, and its lytic enzymes. PBC1 showed a very narrow host range, infecting only 1 of 22 B. cereus strains. Phylogenetic analysis based on the major capsid protein revealed that PBC1 is more closely related to the Bacillus clarkii phage BCJA1c and phages of lactic acid bacteria than to the phages infecting B. cereus. Whole-genome comparison showed that the late-gene region, including the terminase gene, structural genes, and holin gene of PBC1, is similar to that from B. cereus temperate phage 250, whereas their endolysins are different. Compared to the extreme host specificity of PBC1, its endolysin, LysPBC1, showed a much broader lytic spectrum, albeit limited to the genus Bacillus. The catalytic domain of LysPBC1 when expressed alone also showed Bacillus-specific lytic activity, which was lower against the B. cereus group but higher against the Bacillus subtilis group than the full-length protein. Taken together, these results suggest that the virulent phage PBC1 is a useful component of a phage cocktail to control B. cereus, even with its exceptionally narrow host range, as it can kill a strain of B. cereus that is not killed by other phages, and that LysPBC1 is an alternative biocontrol agent against B. cereus. Bacillus cereus is a spore-forming, opportunistic Gram-positive bacterium that is widely distributed in the environment. Because B. cereus produces emetic toxin and enterotoxins, it causes food poisoning, including vomiting and diarrhea, particularly after the consumption of rice-based dishes (1). The Centers for Disease and Control and Prevention (CDC) reported that B. cereus outbreaks account for 2 to 5% of food-borne diseases in the United States (2). In addition to food poisoning, B. cereus is also associated with potentially fatal non-gastrointestinal tract infections due to the various types of toxins, including phospholipases, proteases, and hemolysins, produced during growth (3). For these reasons, there is an urgent need to control B. cereus. Since B. cereus is generally resistant to beta-lactam antibiotics and several strains of B. cereus are also resistant to erythromycin, tetracycline, and fluoroquinolones (1, 4), the demand for alternative methods to control B. cereus has grown (3).Bacteriophages, natural killers of bacteria, have gained increasing attention in the past few decades, particularly in light of emerging antibiotic resistance (5, 6). Bacteriophages are much more specific than antibiotics, so they have minimal impact on commensal bacteria other than the host. Although bacteria can develop resistance to their viral predators, finding a new phage that can kill resistant bacteria is not difficult, because the phage itself continually evolves again...

The PlyB Endolysin of Bacteriophage vB_BanS_Bcp1 Exhibits Broad-Spectrum Bactericidal Activity against Bacillus cereus Sensu Lato Isolates

“…To extend this work and to understand the potential for PlyB as a broadly active therapeutic tool, we examined the lytic activity of PlyB against a wide range of B. cereus sensu lato strains using a spectrophotometry-based lytic assay that monitors the loss of optical density over a 20-min treatment period (indicated as the loss of milliOD 600 [milli-optical density at 600 nm] units per min [ϪmOD/min]). The lytic assay (and associated assays looking at bacterial clearing zones on agar plates) is commonly used to rapidly discern the bacteriolytic activity of endolysins against large target sets of bacterial strains (9,18,(30)(31)(32)(33)(34). Potent lytic activity was demonstrated for PlyB against every B. cereus sensu lato strain tested, including 44 B. cereus sensu stricto, 25 B. thuringiensis, 7 B. mycoides, and 3 B. anthracis strains ( Table 1 and Table S1).…”

“…To pursue a more detailed study of the antimicrobial properties of Bcp1, we now describe the initial identification of Bcp1 from landfill soil (using a B. anthracis reporter strain) and provide a detailed physical characterization showing that Bcp1 is a lytic myovirus with an infective specificity toward a subset of each B. cereus sensu lato species tested, including B. anthracis, B. cereus sensu stricto, B. thuringiensis, and B. mycoides. The broad infectivity of Bcp1 is akin to that of some B. cereus sensu lato phages, including Basilisk (52) and vB_BceM-HSE3 (53), but contrasts with the limited ranges of ␥, Wip1, vB BanS-Tsamsa, and the vB-Bak1, vB-Bak6, and vB-Bak10 phages of B. anthracis (11,(54)(55)(56), as well as the PBC1 and PBC4 phages of B. cereus sensu stricto (18,57). As a lytic bacteriophage with a broad infective range, Bcp1 is appropriate for consideration as a component of a phage cocktail to target B. cereus sensu lato organisms.…”

“…The PlyG endolysin encoded by the ␥ diagnostic phage of B. anthracis, for example, has been shown to exert a potent in vitro bacteriolytic effect against the anthrax agent and is highly efficacious in a mouse model of lethal bacteremia (11). Other endolysins, with various specificities toward the B. cereus sensu lato lineage, have been identified and characterized, including AmiBA2446, LysPBC1, LysBPS13, PlyP56, PlyN74, and PlyTB40 (17)(18)(19)(20).…”

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain

“…A sewage sample was collected from the Seonam Water Reclamation Center in Seoul, South Korea. PBC2 was isolated and purified according to methods previously described (41). Briefly, a 25-ml sample was added to equal volumes of 2ϫ LB broth and incubated with shaking at 37°C for 24 h. After centrifugation (10,000 ϫ g for 10 min), the filtered supernatant was mixed with overnight cultures of a B. cereus host strain for initial propagation.…”

“…Morphological analysis by TEM. Purified PBC2 (1 ϫ 10 10 PFU/ml) was visualized by transmission electron microscopy (TEM) conducted as previously described (41). Based on the morphology, PBC2 was classified according to the guidelines of the International Committee on Taxonomy of Viruses.…”