To explore new approaches of phage-based bio-process of specifically pathogenic Escherichia coli bacteria in food products within a short period. One hundred and forty highly lytic designed coliphages were used. Escherichia coli naturally contaminated and Enterohemorrhagic Escherichia coli experimentally inoculated samples of lettuce, cabbage, meat, and egg were used. In addition, experimentally produced biofilms of E. coli were tested. A phage concentration of 10(3) PFU/ml was used for food products immersion, and for spraying of food products, 10(5) PFU/ml of a phage cocktail was used by applying a 20-s optimal dipping time in a phage cocktail. Food samples were cut into pieces and were either sprayed with or held in a bag immersed in lambda buffer containing a cocktail of 140 phages. Phage bio-processing was successful in eliminating completely E. coli in all processed samples after 48 h storage at 4°C. Partial elimination of E. coli was observed in earlier storage periods (7 and 18 h) at 24° and 37°C. Moreover, E. coli biofilms were reduced >3 log cycles upon using the current phage bio-processing. The use of a phage cocktail of 140 highly lytic designed phages proved highly effective in suppressing E. coli contaminating food products. Proper decontamination/prevention methods of pathogenic E. coli achieved in this study can replace the current chemically less effective decontamination methods.
Colibacillosis is one of the main causes of economic loss in the poultry industry worldwide. Although antibiotics have been used to control this infection, the emergence of antibiotic-resistant bacteria poses a threat to animal and human health. Phage therapy has been reported as one of the potential alternative methods to control bacterial infections. However, efficient phage therapy is highly dependent on the characteristics of the phage isolated. In the present study the characteristics of a lytic phage, ØEC1, which was found to be effective against the causative agent of colibacillosis in chickens in a previous in vivo study, are reported.
The phage N15 protelomerase enzyme (TelN) is essential for the replication of its genome by resolution of its telRL domain, located within a telomerase occupancy site (tos), into hairpin telomeres. Isolation of TelN for in vitro processing of tos, however, is a highly complex process, requiring multiple purification steps. In this study a simplified protocol for crude total protein extraction is described that retains the tos-cleaving activity of TelN for at least 4 weeks, greatly simplifying in vitro testing of its activity. This protocol may be extended for functional analysis of other phage and bacterial proteins, particularly DNA-processing enzymes.
The nucleocapsid protein of Nipah virus produced in Escherichia coli assembled into herringbone-like particles. The amino- and carboxy-termini of the N protein were shortened progressively to define the minimum contiguous sequence involved in capsid assembly. The first 29 aa residues of the N protein are dispensable for capsid formation. The 128 carboxy-terminal residues do not play a role in the assembly of the herringbone-like particles. A region with amino acid residues 30-32 plays a crucial role in the formation of the capsid particle. Deletion of any of the four conserved hydrophobic regions in the N protein impaired capsid formation. Replacement of the central conserved regions with the respective sequences from the Newcastle disease virus restored capsid formation.
Hepatitis B virus-like particles expressed in Escherichia coli were purified using anion exchange adsorbents grafted with polymer poly(oligo(ethylene glycol) methacrylate) in flow-through chromatography mode. The virus-like particles were selectively excluded, while the relatively smaller sized host cell proteins were absorbed. The exclusion of virus-like particles was governed by the accessibility of binding sites (the size of adsorbents and the charge of grafted dextran chains) as well as the architecture (branch-chain length) of the grafted polymer. The branch-chain length of grafted polymer was altered by changing the type of monomers used. The larger adsorbent (90 μm) had an approximately twofold increase in the flow-through recovery, as compared to the smaller adsorbent (30 μm). Generally, polymer-grafted adsorbents improved the exclusion of the virus-like particles. Overall, the middle branch-chain length polymer grafted on larger adsorbent showed optimal performance at 92% flow-through recovery with a purification factor of 1.53. A comparative study between the adsorbent with dextran grafts and the polymer-grafted adsorbent showed that a better exclusion of virus-like particles was achieved with the absorbent grafted with inert polymer. The grafted polymer was also shown to reduce strong interaction between binding sites and virus-like particles, which preserved the particles' structure.
Macrobrachium rosenbergii nodavirus (MrNv) infects giant freshwater prawns and causes white tail disease (WTD). The coding region of the capsid protein of MrNv was amplified with RT-PCR and cloned into the pTrcHis2-TOPO vector. The recombinant plasmid was introduced into Escherichia coli and protein expression was induced with IPTG. SDS-PAGE showed that the recombinant protein containing the His-tag and myc epitope has a molecular mass of about 46 kDa and it was detected by the anti-His antibody in Western blotting. The protein was purified using immobilized metal affinity chromatography (IMAC) and transmission electron microscopic analysis revealed that the recombinant protein assembled into virus-like particles (VLPs) with a diameter of about 30±3 nm. The size of the particles was confirmed by dynamic light scattering. Nucleic acids were extracted from the VLPs and treatment with nucleases showed that they were mainly RNA molecules. This is the first report describing the production of MrNv capsid protein in bacteria and its assembly into VLPs.
Bacteriophage EC1-UPM is an N4-like bacteriophage which specifically infects Escherichia coli O78:K80, an avian pathogenic strain that causes colibacillosis in poultry. The complete genome sequence of bacteriophage EC1-UPM was analysed and compared with other closely related N4-like phage groups to assess their genetic similarities and differences.
The efficacy of bacteriophage EC1, a lytic bacteriophage, against Escherichia coli O78:K80, which causes colibacillosis in poultry, was determined in the present study. A total of 480 one-day-old birds were randomly assigned to 4 treatments groups, each with 4 pens of 30 birds. Birds from the control groups (groups I and II) received PBS (pH 7.4) or 10(10) pfu of bacteriophage EC1, respectively. Group III consisted of birds challenged with 10(8) cfu of E. coli O78:K80 and treated with 10(10) pfu of bacteriophage EC1 at 2 h postinfection, whereas birds from group IV were challenged with 10(8) cfu of E. coli O78:K80 only. All the materials were introduced into the birds by intratracheal inoculation. Based on the results of the present study, the infection was found to be less severe in the treated E. coli-challenged group. Mean total viable cell counts of E. coli identified on eosin methylene blue agar (designated EMB + E. coli) in the lungs were significantly lower in treated, E. coli-challenged birds than in untreated, E. coli-challenged birds on d 1 and 2 postinfection. The EMB + E. coli isolation frequency was also lower in treated birds; no E. coli was detectable in blood samples on any sampling day, and E. coli were isolated only in the liver, heart, and spleen of treated chickens at a ratio of 2/6, 1/6, and 3/6, respectively, at d 1 postinfection. The BW of birds from the E. coli-challenged group treated with bacteriophage EC1 were not significantly different from those of birds from both control groups but were 15.4% higher than those of the untreated, E. coli-challenged group on d 21 postinfection. The total mortality rate of birds during the 3-wk experimental period decreased from 83.3% in the untreated, E. coli-challenged birds (group IV) to 13.3% in birds treated with bacteriophage EC1 (group III). These results suggest that bacteriophage EC1 is effective in vivo and could be used to treat colibacillosis in chickens.
A single chain variable fragment (scFv) specific towards B. pseudomallei exotoxin had previously been generated from an existing hybridoma cell line (6E6AF83B) and cloned into the phage display vector pComb3H. In this study, the scFv was subcloned into the pComb3X vector to facilitate the detection and purification of expressed antibodies. Detection was facilitated by the presence of a hemagglutinin (HA) tag, and purification was facilitated by the presence of a histidine tag. The culture was grown at 30 degrees C until log phase was achieved and then induced with 1 mM IPTG in the absence of any additional carbon source. Induction was continued at 30 degrees C for five h. The scFv was discerned by dual processes-direct enzyme-linked immunosorbent assays (ELISA), and Western blotting. When compared to E. coli strains ER2537 and HB2151, scFv expression was observed to be highest in the E. coli strain Top10F'. The expressed scFv protein was purified via nickel-mediated affinity chromatography and results indicated that two proteins a 52 kDa protein, and a 30 kDa protein were co-purified. These antibodies, when blotted against immobilized exotoxin, exhibited significant specificity towards the exotoxin, compared to other B. pseudomallei antigens. Thus, these antibodies should serve as suitable reagents for future affinity purification of the exotoxin.