Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae

Reduction of Bacterial Load on Broiler Carcasses Using Low-Volume Fluidic Nozzles in Combination with 60 °C Water at 450 Psi Pressure

With the changing regulations in poultry processing, increased pressure is placed on integrators to reduce the number of human enteropathogenic bacteria on the final carcass and/or parts. Reducing the total number of bacteria on broiler carcasses before entering the evisceration side of the processing plant is projected to reduce the number of bacteria on the carcasses after chilling. This study was designed to evaluate the efficacy of a prototype wash cabinet using low volume, fluidic nozzles in combination with high pressure (450 psi) and hot water (60 °C) to remove bacteria from pre-scald, post-scald, or post picked carcasses. Carcasses (n = 5) from each location were obtained from a commercial processing plant, placed into individual sterile sample bags, placed into an insulated container, and transported to the U.S. National Poultry Research Center Pilot Plant within 30 min of collection. Carcasses were hung in standard shackles and sampled pre-wash with pre-moistened, cellulose swabs. All carcasses were washed in the prototype wash cabinet with 60 °C water at 450 psi at a line speed of 52 birds/minute on 15.24 cm centered shackles. Post-wash breast sponge samples were collected identical to pre-wash swabs. Buffered peptone water (BPW) was added, sponges stomached and serially diluted before plating onto total aerobic count (TAC), Enterobacteriaceae (ENT) and Escherichia. coli (EC) Petrifilm® cards. All PetriFilm® cards were incubated at 37 °C for 24 ± 2 h. After incubation, bacterial counts were recorded and converted to log10 CFU/swab. Samples were processed for Campylobacter species using the Tempo® CAM protocol. Four replications were conducted on separate dates. Paired t-tests were used to compare numbers recovered from breast swabs collected before and after the wash cabinet, significance reported at p 10 colony-forming units/carcass (CFU/carcass) for TAC, Ent, and EC Petrifilm®, respectively, and a 2.21 CFU/mL reduction of Campylobacter species using Tempo® CAM. Post-scald, there were significant reductions of 2.09, 1.23, and 0.90 CFU/carcass for TA, Ent, and EC Petrifilm®, respectively, and a 1.14 CFU/mL reduction of Campylobacter species using Tempo® CAM. Post-pick, significant reductions of 0.73, 1.53, and 0.99 CFU/carcass for TA, Ent, and EC Petrifilm®, respectively, and a 0.86 CFU/carcass reduction of Campylobacter species using Tempo® CAM were reported. These data indicate that hot water at high pressure can reduce total bacterial load on carcasses and reduce pathogenic bacteria on carcasses prior to evisceration

A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-l-alanine amidase domain from the endolysin of the thermophilic bacteriophage phi GVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage phi CP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50 degrees C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production

Comparative Genome Maps for Podoviral Bacteriophages Isolated from the Russian Federation and the USA.

<p>The %GC plot displays regions (500-bp window) above and below the average GC content. Open reading frames (ORFs) are depicted as arrows in the predicted direction of transcription and identified putative protein domains are listed in the legend with their respective ORF designation.</p

Whole-genome Similarities as Determined by Tetranucleotide Frequencies.

<p>Dendrogram is based on a dissimilarity matrix for whole bacteriophage genomes constructed in R as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038283#s3" target="_blank">methods</a> section.</p

Phylogenetic Relationships of ΦCPV4, ΦZP2 and ΦCP7R among other related Bacteriophages based on the DNA Polymerase Protein.

<p>Phylogenetic analysis of polymerase amino acid sequences were completed in MEGA5 utilizing MUSCLE for alignments with two thousand bootstrap replications and the <i>Clostridium</i> phage ΦCTP1 polymerase outgroup.</p

Electron Micrograph of Bacteriophage ΦCP7R.

<p>Scale bar represents 100 nm.</p

Shared and Unique Genes present in Bacillus phage Φ29 versus Bacteriophages ΦCPV4, ΦZP2 and ΦCP7R.

<p>Gene clustering was determined by uclust and blastp as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038283#s3" target="_blank">methods</a> section.</p