RESUMEN
This review gathers all, to the best of our current knowledge, known lysins, mainly bacteriophage-derived, that have demonstrated activity against Bacillus anthracis strains. B. anthracis is a spore-forming, toxin-producing bacteria, naturally dwelling in soil. It is best known as a potential biowarfare threat, an etiological agent of anthrax, and a severe zoonotic disease. Anthrax can be treated with antibiotics (ciprofloxacin, penicillin, doxycycline); however, their administration may take up even to 60 days, and different factors can compromise their effectiveness. Bacterial viruses, bacteriophages (phages), are natural enemies of bacteria and use their lytic enzymes, endolysins (lysins), to specifically kill bacterial cells. Harnessing the potential of lysins to combat bacterial infections holds promise for diminishing antibiotic usage and, consequently, addressing the escalating antibiotic resistance in bacteria. In this context, we list the lysins with the activity against B. anthracis, providing a summary of their lytic properties in vitro and the outcomes observed in animal models. Bacillus cereus strain ATCC 4342/RSVF1, a surrogate for B. anthracis, was also included as a target bacteria. KEY POINTS: ⢠More than a dozen different B. anthracis lysins have been identified and studied. ⢠They fall into three blocks regarding their amino acid sequence similarity and most of them are amidases. ⢠Lysins could be used in treating B. anthracis infections.
Asunto(s)
Carbunco , Antibacterianos , Bacillus anthracis , Endopeptidasas , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/virología , Carbunco/tratamiento farmacológico , Carbunco/microbiología , Animales , Endopeptidasas/farmacología , Endopeptidasas/metabolismo , Endopeptidasas/genética , Antibacterianos/farmacología , Bacteriófagos/genética , Bacillus cereus/efectos de los fármacos , Bacillus cereus/virología , Humanos , Fagos de Bacillus/genéticaRESUMEN
The common cooccurrence of antibiotics and phages in both natural and engineered environments underscores the need to understand their interactions and implications for bacterial control and antibiotic resistance propagation. Here, aminoglycoside antibiotics that inhibit protein synthesis (e.g., kanamycin and neomycin) impeded the replication of coliphage T3 and Bacillus phage BSP, reducing their infection efficiency and mitigating their hindrance of bacterial growth, biofilm formation, and tolerance to antibiotics. For example, treatment with phage T3 reduced subsequent biofilm formation by Escherichia coli liquid cultures to 53% ± 5% of that of the no-phage control, but a smaller reduction of biofilm formation (89% ± 10%) was observed for combined exposure to phage T3 and kanamycin. Despite sharing a similar mode of action with aminoglycosides (i.e., inhibiting protein synthesis) and antagonizing phage replication, albeit to a lesser degree, tetracyclines did not inhibit bacterial control by phages. Phage T3 combined with tetracycline showed higher suppression of biofilm formation than when combined with aminoglycosides (25% ± 6% of the no-phage control). The addition of phage T3 to E. coli suspensions with tetracycline also suppressed the development of tolerance to tetracycline. However, this suppression of antibiotic tolerance development disappeared when tetracycline was replaced with 3 mg/liter kanamycin, corroborating the greater antagonism with aminoglycosides. Overall, this study highlights this overlooked antagonistic effect on phage proliferation, which may attenuate phage suppression of bacterial growth, biofilm formation, antibiotic tolerance, and maintenance of antibiotic resistance genes. IMPORTANCE The coexistence of residual antibiotics and phages is common in many environments, which underscores the need to understand their interactive effects on bacteria and the implications for antibiotic resistance propagation. Here, aminoglycosides acting as bacterial protein synthesis inhibitors impeded the replication of various phages. This alleviated the suppressive effects of phages against bacterial growth and biofilm formation and diminished bacterial fitness costs that suppress the emergence of tolerance to antibiotics. We show that changes in bacteria caused by environmentally relevant concentrations of sublethal antibiotics can affect phage-host dynamics that are commonly overlooked in vitro but can result in unexpected environmental consequences.
Asunto(s)
Antibacterianos/farmacología , Fagos de Bacillus/efectos de los fármacos , Bacillus cereus/efectos de los fármacos , Bacteriófago T3/efectos de los fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Kanamicina/farmacología , Neomicina/farmacología , Fagos de Bacillus/crecimiento & desarrollo , Bacillus cereus/fisiología , Bacillus cereus/virología , Bacteriófago T3/crecimiento & desarrollo , Biopelículas/crecimiento & desarrollo , Escherichia coli/fisiología , Escherichia coli/virología , Tetraciclina/farmacologíaRESUMEN
A new Bacillus cereus phage, SWEP1, was isolated from black soil. The host lysis activity of phage SWEP1 has a relatively short latent time (20 min) and a small burst size of 83 PFU. The genome of SWEP1 consists of 162,461 bp with 37.77% G+C content. The phage encodes 278 predicted proteins, 103 of which were assigned functionally. No tRNA genes were found. Comparative genomics analysis indicated that SWEP1 is related to Bacillus phage B4 (86.91% identity, 90% query coverage). Phenotypic and genotypic characterization suggested that SWEP1 is a new member of a new species in the genus Bequatrovirus, family Herelleviridae.
Asunto(s)
Fagos de Bacillus/fisiología , Bacillus cereus/virología , Genoma Viral , Fagos de Bacillus/genética , Fagos de Bacillus/aislamiento & purificación , Bacillus cereus/aislamiento & purificación , Composición de Base , Replicación del ADN/genética , Genómica , Genotipo , Microscopía Electrónica de Transmisión , Filogenia , ARN de Transferencia/genética , Microbiología del Suelo , TemperaturaRESUMEN
As the most abundant biological entities, viruses are major players in marine ecosystems. However, our knowledge about virus-host interactions and viral ecology in the deep sea remains very limited. In this study, a novel bacteriophage (designated as phage BVE2) infecting Bacillus cereus group bacteria, was isolated from deep-sea sediments. Phage BVE2 caused host lysis within 1.5 h after infection. However, the presence of two integrase-encoding genes in the BVE2 genome suggested that BVE2 may also follow a temperate strategy. The genome of phage BVE2 is approximately 20 kb in length and is predicted to encode 28 proteins. Genomic and phylogenetic analysis suggested that BVE2 is a highly mosaic phage that has inherited genetic features from Wbeta-like viruses, B. cereus prophages, and its host, suggesting that frequent horizontal gene transfer events occurred during its evolution. This study will help to reveal the evolutionary history of Wbeta-like viruses and improve our understanding of viral diversity and virus-host interactions in the deep sea.
Asunto(s)
Fagos de Bacillus/clasificación , Fagos de Bacillus/aislamiento & purificación , Bacillus cereus/virología , Genoma Viral , Agua de Mar/virología , Fagos de Bacillus/genética , Fagos de Bacillus/crecimiento & desarrollo , Bacteriólisis , Transferencia de Gen Horizontal , Genes Bacterianos , Lisogenia , Recombinación Genética , Análisis de Secuencia de ADNRESUMEN
Lytic bacteriophages (or phages) drive bacterial mortality by elaborating exquisite abilities to bind, breach, and destroy bacterial cell membranes and subjugate critical bacterial cell functions. These antimicrobial activities make phages ideal candidates to serve as, or provide sources of, biological control measures for bacterial pathogens. In this study, we isolated the Myoviridae phage vB_BanS_Bcp1 (here referred to as Bcp1) from landfill soil, using a Bacillus anthracis host. The antimicrobial activities of both Bcp1 and its encoded endolysin, PlyB, were examined across different B. cereussensu lato group species, including B. cereussensu stricto, Bacillus thuringiensis, and Bacillus anthracis, with pathogenic potential in humans and multiple different uses in biotechnological applications. The Bcp1 phage infected only a subset (11 to 66%) of each B. cereussensu lato species group tested. In contrast, functional analysis of purified PlyB revealed a potent bacteriolytic activity against all B. cereussensu lato isolates tested (n = 79). PlyB was, furthermore, active across broad temperature, pH, and salt ranges, refractory to the development of resistance, bactericidal as a single agent, and synergistic with a second endolysin, PlyG. To confirm the potential for PlyB as an antimicrobial agent, we demonstrated the efficacy of a single intravenous treatment with PlyB alone or combination with PlyG in a murine model of lethal B. anthracis infection. Overall, our findings show exciting potential for the Bcp1 bacteriophage and the PlyB endolysin as potential new additions to the antimicrobial armamentarium.IMPORTANCE Organisms of the Bacillus cereussensu lato lineage are ubiquitous in the environment and are responsible for toxin-mediated infections ranging from severe food poisoning (B. cereussensu stricto) to anthrax (Bacillus anthracis). The increasing incidence of many of these infections, combined with the specter of antibiotic resistance, has created a need for novel antimicrobials with potent activity, including bacteriophages (or phages) and phage-encoded products (i.e., endolysins). In this study, we describe a broadly infective phage, Bcp1, and its encoded endolysin, PlyB, which exhibited a rapidly bacteriolytic effect against all B. cereussensu lato isolates tested with no evidence of evolving resistance. Importantly, PlyB was highly efficacious in a mouse model of lethal bacteremia with B. anthracis Both the Bcp1 phage and the PlyB endolysin represent novel mechanisms of action compared to antibiotics, with potential applications to address the evolving problem of antimicrobial resistance.
Asunto(s)
Antibacterianos/farmacología , Fagos de Bacillus/fisiología , Bacillus anthracis/efectos de los fármacos , Bacillus cereus/virología , Endopeptidasas/farmacología , Myoviridae/fisiología , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Microbiología del Suelo , Instalaciones de Eliminación de ResiduosRESUMEN
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.
Asunto(s)
Fagos de Bacillus/enzimología , Bacillus cereus/virología , Dominio Catalítico , Endopeptidasas/metabolismo , Esporas Bacterianas/virología , Antiinfecciosos , Fagos de Bacillus/genética , Fagos de Bacillus/aislamiento & purificación , Bacillus cereus/metabolismo , Pared Celular/metabolismo , Endopeptidasas/química , Endopeptidasas/genética , Especificidad del Huésped , Modelos Moleculares , Peptidoglicano/metabolismo , Mutación Puntual , Conformación Proteica , Dominios Proteicos/genética , Alineación de Secuencia , Esporas Bacterianas/metabolismoRESUMEN
In the study, three Bacillus cereus-specific phages, named DK1, DK2 and DK3, belonging to the family Podoviridae, were isolated from Pearl River water and sludge in Guangzhou, China. The genomes of DK1, DK2 and DK3 were 27,180 bp, 26,357 bp, and 26,865 bp in length and contained 49, 45 and 46 open reading frames, respectively. Among the three phages, DK2 shared the highest genome sequence similarity (96% identity) with DK3. Genes encoding rRNA, tRNA, virulence factors and antibiotic resistance were absent in these phage genomes. In addition, comparative genomic and phylogenetic analysis revealed that they were novel phages of B. cereus. Each genome encoded a putative endolysin that might be of value for the control of the foodborne pathogen B. cereus.
Asunto(s)
Bacillus cereus/virología , Genoma Viral/genética , Podoviridae/genética , Podoviridae/aislamiento & purificación , Ríos/virología , Aguas del Alcantarillado/virología , China , Endopeptidasas/genética , Sistemas de Lectura Abierta , Podoviridae/clasificaciónRESUMEN
The arm race between bacteria and phages leads to the development of many phage resistance mechanisms. Bacteria are perpetually changing their strategy to avert phage infection and killing. Recently, a study published that shed light on a novel bacterial defense system called bacteriophage exclusion (BREX). BREX system is a six-gene cassette in Bacillus cereus which provides complete phage resistance to a broad range of phages, including lytic and temperate ones. It is a novel defense strategy which involves DNA methylation of the host cell and block phage DNA replication.
Asunto(s)
Bacillus cereus/virología , Bacillus subtilis/virología , Bacteriófagos/patogenicidad , Bacillus cereus/genética , Bacillus cereus/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Sistemas CRISPR-Cas , Metilación de ADN , ADN Viral/biosíntesis , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Replicación ViralRESUMEN
Bacterial endospores can serve as phage genome protection shells against various environmental stresses to enhance microbial control applications. The genomes of polyvalent lytic Bacillus phages PBSC1 and PBSC2, which infect both B. subtilis subsp. subtilis and B. cereus NRS 248, were incorporated into B. subtilis endospores (without integration into the host chromosome). When PBSC1 and PBSC2 were released from germinating endospores, they significantly inhibited the growth of the targeted opportunistic pathogen B. cereus Optimal endospore entrapment was achieved when phages were introduced to the fast-sporulating prespores at a multiplicity of infection of 1. Longer endospore maturation (48 h versus 24 h) increased both spore yield and efficiency of entrapment. Compared with free phages, spore-protected phage genomes showed significantly higher resistance toward high temperatures (60 to 80°C), extreme pH (pH 2 or pH 12), and copper ions (0.1 to 10 mg/liter). Endospore germination is inducible by low concentrations of l-alanine or by a germinant mixture (l-asparagine, d-glucose, d-fructose, and K+) to trigger the expression, assembly, and consequent release of phage particles within 60 to 90 min. Overall, the superior resiliency of polyvalent phages protected by endospores might enable nonrefrigerated phage storage and enhance phage applications after exposure to adverse environmental conditions.IMPORTANCE Bacteriophages are being considered for the control of multidrug-resistant and other problematic bacteria in environmental systems. However, the efficacy of phage-based microbial control is limited by infectivity loss during phage delivery and/or storage. Here, we exploit the pseudolysogenic state of phages, which involves incorporation of their genome into bacterial endospores (without integration into the host chromosome), to enhance survival in unfavorable environments. We isolated polyvalent (broad-host-range) phages that efficiently infect both benign and opportunistically pathogenic Bacillus strains and encapsulated the phage genomes in B. subtilis endospores to significantly improve resistance to various environmental stressors. Encapsulation by spores also significantly enhanced phage genome viability during storage. We also show that endospore germination can be induced on demand with nutrient germinants that trigger the release of active phages. Overall, we demonstrate that encapsulation of polyvalent phage genomes into benign endospores holds great promise for broadening the scope and efficacy of phage biocontrol.
Asunto(s)
Fagos de Bacillus/genética , Bacillus cereus/virología , Bacillus subtilis/virología , Genoma Viral , Esporas Bacterianas/virología , Fagos de Bacillus/química , Fagos de Bacillus/crecimiento & desarrollo , Bacillus cereus/genética , Bacillus cereus/crecimiento & desarrollo , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Calor , Concentración de Iones de Hidrógeno , Esporas Bacterianas/química , Esporas Bacterianas/genética , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
OBJECTIVE: Infants with a weak immune system are prone to infection with Bacillus cereus, which is commonly found in natural environments. With the aim of achieving better control of this pathogenic bacterium, in the present study we characterized a new bacteriophage, ΦBC01. METHODS: Bacteriophage particles were analyzed by transmission electron microscopy. Factors influencing adsorption were identified in a double-layer plaque assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted to determine the size of major structural proteins. The complete genome of the phage was analyzed. RESULTS: Bacteriophage particles (105.3 ± 3.1 nm icosahedral head and 208.8 ± 3.6 nm contractile tail) were identified as Myoviridae. ΦBC01 was found to have host specificity to B. cereus. Major structural proteins of ΦBC01 showed 2 well-pronounced bands of 99 and 56 kDa. The 158,385-bp genome sequence of ΦBC01 was determined: 56 of the 239 open reading frames were annotated, indicating involvement in bacteriophage DNA manipulation, cell lysis, packaging, virion structure, and other functions. CONCLUSION: Because of characterization and genotyping of a new bacteriophage from soil samples containing earthworms, this study provides a baseline for the development of alternatives to antibiotics.
Asunto(s)
Fagos de Bacillus/genética , Bacillus cereus/virología , Genoma Viral , Genómica , Secuenciación Completa del Genoma , Fagos de Bacillus/ultraestructura , Genómica/métodos , Especificidad del Huésped , Filogenia , Microbiología del SueloRESUMEN
Bacteriophage Deep-Purple, isolated from an agricultural soil in Belgium, lyses the emetic Bacillus weihenstephanensis strain LH002 and exhibits a lytic activity against 55% of emetic Bacillus cereus and B. weihenstephanensis strains. Deep-Purple is able to complete its lytic cycle within 45 min and is stable to a large range of pHs and temperatures below 60 °C. It possesses an icosahedral head of about 63 nm in diameter and a non-contractile tail of approximately 165 nm in length. The genome of this newly classifiable Siphoviridae family member is 36,278 bp long, with a G+C content of 38.36% and 40 putative CDSs. Most CDSs do not display similarity with other B. cereus group phages supporting the idea that Deep-Purple belongs to a new and currently uncharacterised Siphoviridae subfamily.
Asunto(s)
Fagos de Bacillus/genética , Fagos de Bacillus/aislamiento & purificación , Bacillus cereus/virología , Genoma Viral , Siphoviridae/genética , Siphoviridae/aislamiento & purificación , Fagos de Bacillus/clasificación , Composición de Base , Bélgica , Filogenia , Siphoviridae/clasificación , Microbiología del Suelo , Secuenciación Completa del GenomaRESUMEN
Bacillus cereus is widely distributed food-borne pathogenic bacterium. Due to the harmness to human hearth and the generation of multidrug-resistant B. cereus, it is urgent to develop novel antimicrobial agents. Phage and phage endolysin were taken as novel antimicrobial substance for their specific lytic activity against pathogenic bacteria. In this study, a Myoviridae family phage, designated as vB_BceM-HSE3, infecting the pathogenic multidrug-resistant B. cereus strain was isolated and characterized along with its endolysin. Phage vB_BceM-HSE3 can specially infect the B. cereus group strains, including B. cereus, B. anthracis, and B. thuringiensis, and exhibits high temperature and pH tolerance, which endow it with high potential for been used in controlling pathogenic B. cereus group strains. Genomic analysis reveals that vB_BceM-HSE3 is a novel phage and only shows extremely low genome similarity with available phage genome. Functional analysis of endolysin PlyHSE3 encoding by vB_BceM-HSE3 shows that PlyHSE3 exhibits broader lytic spectrum than the phage and can lyse all the tested B. cereus group strains as well as the tested pathogenic strain of P. aeruginosa. PlyHSE3 also shows broad temperature and pH tolerance, and can efficiently lyse B. cereus strain at temperature at 4 °C and higher than 45 °C, which indicating that PlyHSE3 might can be used in controlling food-borne B. cereus during both the cold storage of food and the stage after the heat treatment of food. The findings of this study enrich our understanding of phage diversity as well as providing resources for developing phage therapy.
Asunto(s)
Bacillus cereus/efectos de los fármacos , Bacillus cereus/virología , Bacteriófagos/fisiología , Farmacorresistencia Microbiana , Endopeptidasas/metabolismo , Bacillus cereus/metabolismo , Bacteriófagos/metabolismo , Calor , Concentración de Iones de HidrógenoRESUMEN
BACKGROUND: Most tailed bacteriophages (phages) feature linear dsDNA genomes. Characterizing novel phages requires an understanding of complete genome sequences, including the definition of genome physical ends. RESULT: We sequenced 48 Bacillus cereus phage isolates and analyzed Next-generation sequencing (NGS) data to resolve the genome configuration of these novel phages. Most assembled contigs featured reads that mapped to both contig ends and formed circularized contigs. Independent assemblies of 31 nearly identical I48-like Bacillus phage isolates allowed us to observe that the assembly programs tended to produce random cleavage on circularized contigs. However, currently available assemblers were not capable of reporting the underlying phage genome configuration from sequence data. To identify the genome configuration of sequenced phage in silico, a terminus prediction method was developed by means of 'neighboring coverage ratios' and 'read edge frequencies' from read alignment files. Termini were confirmed by primer walking and supported by phylogenetic inference of large DNA terminase protein sequences. CONCLUSIONS: The Terminus package using phage NGS data along with the contig circularity could efficiently identify the proximal positions of phage genome terminus. Complete phage genome sequences allow a proposed characterization of the potential packaging mechanisms and more precise genome annotation.
Asunto(s)
Fagos de Bacillus/genética , Bacillus cereus/virología , Genoma Viral , Secuencia de Bases , Mapeo Cromosómico , Mapeo Contig , ADN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Análisis de Secuencia de ADN , Proteínas Virales/genéticaRESUMEN
The cold-active bacteriophage VMY22, belonging to the Podoviridae family, was isolated from Mingyong Glacier in China. Sequence analysis revealed that the genome is 18,609 bp long, with an overall G + C content of 36.4 mol%, and 25 open reading frames (ORFs). The sequence contains 46 potential promoters, 6 transcription terminators, and no tRNAs. Most of the ORFs show a high degree of similarity to B103 (NC_004165). Two noteworthy findings were made. First, one of the predicted proteins, ORF 19, shows high sequence similarity to the bacteriocin biosynthesis protein from Bacillus cereus. From this information, we propose that the VMY22 phage is at an intermediate phase in its coevolution with its bacterial host. Second, seven of the hypothetical proteins appear to be unique to this cold-active B. cereus phage (i.e., not found in temperate-active B. cereus phages). These observations add to our current knowledge about the coevolution of bacteriophages and their hosts. The identification of a novel group of gene and protein structures and functions will lead to a better understanding of cold-adaptation mechanisms in bacteria and their bacteriophages.
Asunto(s)
Fagos de Bacillus/química , Fagos de Bacillus/genética , Bacillus cereus/virología , ADN Viral/química , ADN Viral/genética , Genoma Viral , Antibacterianos/química , Fagos de Bacillus/clasificación , Fagos de Bacillus/aislamiento & purificación , Proteínas Bacterianas/química , Composición de Base , China , Mapeo Cromosómico , Evolución Molecular , Sistemas de Lectura Abierta , Filogenia , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Terminación de la Transcripción Genética , Proteínas Virales/químicaRESUMEN
This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages suggested that JL and Shanette were members of the family Myoviridae, which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis, and B. thuringiensis. This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria. Importance: The Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein we provide the results of detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with results of genomic, proteomic, and structural analyses, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.
Asunto(s)
Fagos de Bacillus/genética , Fagos de Bacillus/metabolismo , Bacillus cereus/virología , Genoma Bacteriano , Proteoma , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Sistemas de Lectura Abierta , VirulenciaRESUMEN
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.
Asunto(s)
Antiinfecciosos/metabolismo , Fagos de Bacillus/enzimología , Fagos de Bacillus/crecimiento & desarrollo , Bacillus cereus/efectos de los fármacos , Bacillus cereus/crecimiento & desarrollo , Bacteriólisis/efectos de los fármacos , Endopeptidasas/metabolismo , Fagos de Bacillus/clasificación , Fagos de Bacillus/aislamiento & purificación , Bacillus cereus/virología , Proteínas de la Cápside/genética , Análisis por Conglomerados , ADN Viral/química , ADN Viral/genética , Orden Génico , Genoma Viral , Especificidad del Huésped , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Siphoviridae/clasificación , Siphoviridae/enzimología , Siphoviridae/crecimiento & desarrollo , Siphoviridae/aislamiento & purificación , SinteníaRESUMEN
Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus.
Asunto(s)
Bacillus cereus/aislamiento & purificación , Bacteriófagos/enzimología , Endopeptidasas/química , Secuencia de Aminoácidos , Bacillus cereus/virología , Sitios de Unión , Técnicas Biosensibles/métodos , Pared Celular/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Bacteriophage JBP901, isolated from fermented food, is specific for Bacillus cereus group species and exhibits a broad host spectrum among a large number of B. cereus isolates. Genome sequence analysis revealed a linear 159,492-bp genome with overall G+C content of 39.7 mol%, and 201 ORFs. The presence of a putative methylase, the large number of tRNAs, and the large number of nucleotide-metabolism- and replication-related genes in JBP901 reflects its broad lytic capacity. Most of the ORFs showed a high degree of similarity to Bcp1, Bc431v3 and BCP78, and various comparative genomics analyses also consistently clustered JBP901 with orphan (unclassified) Bacillus phages in the subfamily Spounavirinae of the family Myoviridae, supporting the presence of a distinguishable group in the subfamily.
Asunto(s)
Fagos de Bacillus/genética , Fagos de Bacillus/aislamiento & purificación , Bacillus cereus/virología , ADN Viral/química , ADN Viral/genética , Genoma Viral , Fagos de Bacillus/fisiología , Bacillus cereus/aislamiento & purificación , Composición de Base , Análisis por Conglomerados , Microbiología de Alimentos , Genes Virales , Especificidad del Huésped , Datos de Secuencia Molecular , Myoviridae/genética , Myoviridae/aislamiento & purificación , Myoviridae/fisiología , Sistemas de Lectura Abierta , Filogenia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Bacillus cereus group-specific bacteriophage BCP8-2 exhibits a broad lysis spectrum among food and human isolates (330/364) of B. cereus while not infecting B. subtilis (50) or B. licheniformis (12) strains. Its genome is 159,071 bp long with 220 open reading frames, including genes for putative methyltransferases, metallo-beta-lactamase, and a sporulation-related SpoIIIE homolog, as wells as 18 tRNAs. Comparative genome analysis showed that BCP8-2 is related to the recently proposed Bastille-like phages, but not with either SPO1-like or Twort-like phages of the subfamily Spounavirinae.
Asunto(s)
Fagos de Bacillus/genética , Fagos de Bacillus/aislamiento & purificación , Bacillus cereus/virología , Genoma Viral , Myoviridae/genética , Myoviridae/aislamiento & purificación , Proteínas Virales/genética , beta-Lactamasas/genética , Fagos de Bacillus/clasificación , Fagos de Bacillus/enzimología , Secuencia de Bases , Datos de Secuencia Molecular , Myoviridae/clasificación , Myoviridae/enzimología , Sistemas de Lectura Abierta , Filogenia , Proteínas Virales/metabolismo , beta-Lactamasas/metabolismoRESUMEN
The study of extremophilic phages may reveal new phage families as well as different mechanisms of infection, propagation and lysis to those found in phages from temperate environments. We describe a novel siphovirus, GVE3, which infects the thermophile Geobacillus thermoglucosidasius. The genome size is 141,298 bp (G+C 29.6%), making it the largest Geobacillus spp-infecting phage known. GVE3 appears to be most closely related to the recently described Bacillus anthracis phage vB_BanS_Tsamsa, rather than Geobacillus-infecting phages described thus far. Tetranucleotide usage deviation analysis supports this relationship, showing that the GVE3 genome sequence correlates best with B. anthracis and Bacillus cereus genome sequences, rather than Geobacillus spp genome sequences.