RESUMEN
This study focused on the isolation and characterization of bacteriophages with specific activity against toxin-producing and multidrug-resistant strains of Bacillus cereus sensu stricto (B. cereus s. s.). Ten different samples yielded six bacteriophages by utilizing the double-layer agar technique. The most promising phage, vB_BceS-M2, was selected based on its broad host range and robust lytic activity against various B. cereus s. s. strains. The phage vB_BceS-M2 had a circular double-stranded DNA genome of 56,482 bp. This phage exhibited stability over a wide range of temperatures and pH values, which is crucial for its potential application in food matrices. The combined effect of phage vB_BceS-M2 and nisin, a widely used antimicrobial peptide, was investigated to enhance antimicrobial efficacy against B. cereus in food. The results suggested that nisin showed synergy and combined effect with the phage, potentially overcoming the growth of phage-resistant bacteria in the broth. Furthermore, practical applications were conducted in various liquid and solid food matrices, including whole and skimmed milk, boiled rice, cheese, and frozen meatballs, both at 4 and 25 °C. Phage vB_BceS-M2, either alone or in combination with nisin, reduced the growth rate of B. cereus in foods other than whole milk. The combination of bacteriophage and nisin showed promise for the development of effective antimicrobial interventions to counteract toxigenic and antibiotic-resistant B. cereus in food.
Asunto(s)
Antibacterianos , Bacillus cereus , Farmacorresistencia Bacteriana Múltiple , Microbiología de Alimentos , Nisina , Antibacterianos/farmacología , Bacillus cereus/virología , Bacillus cereus/efectos de los fármacos , Fagos de Bacillus/genética , Bacteriófagos , Queso/microbiología , Concentración de Iones de Hidrógeno , Leche/microbiología , Nisina/farmacología , Oryza/microbiología , TemperaturaRESUMEN
Anthrax is an acute infectious zoonotic disease caused by Bacillus anthracis, a bacterium that is considered a potential biological warfare agent. Bacillus bacteriophages shape the composition and evolution of bacterial communities in nature and therefore have important roles in the ecosystem community. B. anthracis phages are not only used in etiological diagnostics but also have promising prospects in clinical therapeutics or for disinfection in anthrax outbreaks. In this study, two temperate B. anthracis phages, vB_BanS_A16R1 (A16R1) and vB_BanS_A16R4 (A16R4), were isolated and showed siphovirus-like morphological characteristics. Genome sequencing showed that the genomes of phages A16R1 and A16R4 are 36,569 bp and 40,059 bp in length, respectively. A16R1 belongs to the genus Wbetavirus, while A16R4 belongs to the genus Hubeivirus and is the first phage of that genus found to lyse B. anthracis. Because these two phages can comparatively specifically lyse B. anthracis, they could be used as alternative diagnostic tools for identification of B. anthracis infections.
Asunto(s)
Fagos de Bacillus , Bacillus anthracis , Genoma Viral , Bacillus anthracis/virología , Genoma Viral/genética , Fagos de Bacillus/aislamiento & purificación , Fagos de Bacillus/genética , Fagos de Bacillus/clasificación , Siphoviridae/genética , Siphoviridae/aislamiento & purificación , Siphoviridae/clasificación , FilogeniaRESUMEN
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
Bacillus cereus is a foodborne pathogen that induces vomiting and diarrhea in affected individuals. It exhibits resistance to traditional sterilization methods and has a high contamination rate in dairy products and rice. Therefore, the development of a new food safety controlling strategy is necessary. In this research, we isolated and identified a novel phage named vB_BceP_LY3, which belongs to a new genus of the subfamily Northropvirinae. This phage demonstrates a short latency period and remains stable over a wide range of temperatures (4-60 °C) and pH levels (4-11). The 28,124 bp genome of LY3 does not contain any antibiotic-resistance genes or virulence factors. With regards to its antibacterial properties, LY3 not only effectively inhibits the growth of B. cereus in TSB (tryptic soy broth), but also demonstrates significant inhibitory effects in various food matrices. Specifically, LY3 treatment at 4 °C with a high MOI (MOI = 10,000) can maintain B. cereus levels below the detection limit for up to 24 h in milk. LY3 represents a safe and promising biocontrol agent against B. cereus, possessing long-term antibacterial capabilities and stability.
Asunto(s)
Bacillus cereus , Microbiología de Alimentos , Leche , Oryza , Oryza/microbiología , Bacillus cereus/virología , Leche/microbiología , Animales , Genoma Viral , Contaminación de Alimentos/prevención & control , Contaminación de Alimentos/análisis , Fagos de Bacillus/genética , Fagos de Bacillus/aislamiento & purificación , Fagos de Bacillus/clasificación , Fagos de Bacillus/fisiología , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiologíaRESUMEN
Bacillus subtilis is a Gram-positive bacterium that is widely used in fermentation and in the pharmaceutical industry. Phage contamination occasionally occurs in various fermentation processes and causes significant economic loss. Here, we report the isolation and characterization of a temperate B. subtilis phage, termed phi18-2, from spore powder manufactured in a fermentation plant. Transmission electron microscopy showed that phi18-2 has a symmetrical polyhedral head and a long noncontractile tail. Receptor analysis showed that phi18-2 recognizes wall teichoic acid (WTA) for infection. The phage virions have a linear double-stranded DNA genome of 64,467 bp with identical direct repeat sequences of 309 bp at each end of the genome. In lysogenic cells, the phage genome was found to be present in the cytoplasm without integration into the host cell chromosome, and possibly as a linear phage-plasmid with unmodified ends. Our data may provide some insight into the molecular basis of the unique lysogenic cycle of phage phi18-2.
Asunto(s)
Fagos de Bacillus , Bacteriófagos , Bacteriófagos/genética , Fagos de Bacillus/genética , ADN Viral/genética , Lisogenia , Genoma Viral , Plásmidos/genética , CitoplasmaRESUMEN
Bacillus spp. are often used as probiotics; however, they can be infected by phages, leading to significant economic losses. Biocidal and thermal treatments are considered rapid and effective methods for controlling microbial contamination. To prevent viral contamination in industrial dairy production, the impact of temperature and biocides on the viability of Bacillus methylotrophic phage BM-P1 was assessed. The results demonstrated that reconstituted skim milk (RSM) as a medium showed the most effective protective effect on phage BM-P1. Treatment at 90°C for 5 min or 72°C for 10 min inactivated it to nondetectable levels from the initial titer of 7.19 ± 0.11 log, regardless of the culture medium. Sodium hypochlorite exhibited the best inactivating effect, which could reduce the phage titer below the detection level in 4 min at 50 ppm. Additionally, treatment with 75% ethanol for 20 min or 50% isopropanol for 30 min could achieve inactivation to nondetectable levels. The inactivating effect of peracetic acid was limited; even when treated at the highest concentration (0.45%) for 60 min, only a 2.47 ± 0.17 log reduction was observed. This study may provide some theoretical basis and data support for establishing measures against Bacillus spp. phages.
Asunto(s)
Fagos de Bacillus , Bacillus , Desinfectantes , Bacteriófago P1 , Calor , Desinfectantes/farmacologíaRESUMEN
The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein-DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.
Asunto(s)
Fagos de Bacillus , Bacillus subtilis , Fagos de Bacillus/genética , Fagos de Bacillus/química , Bacillus subtilis/virología , Replicación del ADN , ADN Viral/genética , Nucleoproteínas/metabolismo , Proteínas Virales/metabolismoRESUMEN
Phages can use a small-molecule communication arbitrium system to coordinate lysis-lysogeny decisions, but the underlying mechanism remains unknown. Here we determined that the arbitrium system in Bacillus subtilis phage phi3T modulates the bacterial toxin-antitoxin system MazE-MazF to regulate the phage life cycle. We show that phi3T expresses AimX and YosL, which bind to and inactivate MazF. AimX also inhibits the function of phi3T_93, a protein that promotes lysogeny by binding to MazE and releasing MazF. Overall, these mutually exclusive interactions promote the lytic cycle of the phage. After several rounds of infection, the phage-encoded AimP peptide accumulates intracellularly and inactivates the phage antiterminator AimR, a process that eliminates aimX expression from the aimP promoter. Therefore, when AimP increases, MazF activity promotes reversion back to lysogeny, since AimX is absent. Altogether, our study reveals the evolutionary strategy used by arbitrium to control lysis-lysogeny by domesticating and fine-tuning a phage-defence mechanism.
Asunto(s)
Fagos de Bacillus , Lisogenia , Fagos de Bacillus/fisiología , Péptidos/metabolismo , Muerte CelularRESUMEN
Temperate Bacillus phages often utilize arbitrium communication to control lysis/lysogeny decisions, but the mechanisms by which this control is exerted remains largely unknown. Here we find that the arbitrium system of Bacillus subtilis phage Ï3T modulates the host-encoded MazEF toxin-antitoxin system to this aim. Upon infection, the MazF ribonuclease is activated by three phage genes. At low arbitrium signal concentrations, MazF is inactivated by two phage-encoded MazE homologues: the arbitrium-controlled AimX and the later-expressed YosL proteins. At high signal, MazF remains active, promoting lysogeny without harming the bacterial host. MazF cleavage sites are enriched on transcripts of phage lytic genes but absent from the phage repressor in Ï3T and other Spß-like phages. Combined with low activation levels of MazF during infections, this pattern explains the phage-specific effect. Our results show how a bacterial toxin-antitoxin system has been co-opted by a phage to control lysis/lysogeny decisions without compromising host viability.