Isolation, Propagation and Biocontrol Activity of Indigenous Bacteriophages against Brucella abortus

Brucellosis, being a zoonotic disease, is difficult to control despite the availability of vaccines. Bovine brucellosis could be controlled effectively using brucellaphages. This study was conducted to isolate bacteriophages against Brucella abortus from cattle farms sewage/slurry samples (n=50). Isolation and propagation of brucellaphages was made through spot and plaque assay. Two samples (n=2) were found positive on the plates as circular, clear, and pinpoint plaques (0.3 to 0.5 mm) with 4.6×106 PFU/mL of brucellaphage titre against Brucella abortus RB51. One- step growth experiments revealed latent period of 120 min and burst size of 93 and 114 PFU for two isolated brucellaphages (ϕP1 and ϕP2) respectively. These phages were unable to lyse Staphylococcus aureus, Streptococcus spp., Salmonella spp., Escherichia coli, Bacillus subtilis and Pasteurella multocida. Isolated brucellaphages were stable up to 60°C and between 7 to 9 pH. No loss in phage titres were observed at 4°C but phage titres were reduced by one log at (-20°C) overnight. There was no effect of SM buffer, normal saline and EDTA on stability of brucellaphages and addition of divalent salts in medium showed significant increase in PFU. However, treatment with SDS and chloroform destroyed the phages in one hour exposure. The phages were found carrying double stranded DNA (~40 kb size) and two prominent protein bands of 45 kDa and 70 kDa. Biocontrol activity of brucellaphages showed average Brucella abortus count reduction to 4.5×103 from 5.0×108 CFU/ g of soil sample within 48 h when treated with maximum phage titre of 5.0×1011 PFU/ mL. Hence, this study revealed that brucellaphages are present in our environment and can potentially be consider for their cost-effective practical applications in inactivation of Brucella abortus after comprehensive experimental evaluations. © 2021 Friends Science Publishers

Improving phage titre through examining point of infection

Bacteriophages are viruses that cause the lysis of bacteria. They have recently been used to combat antimicrobial resistant infections as an alternative therapy to antibiotics. Their production and propagation, however, remains understudied and will be key to obtaining titres required for future clinical studies and research. Previous work suggests that temperature of infection significantly influences the production process and output yield of phage, with a reduction in temperature from 37ºC to 28ºC resulting in significant increases in productivity for multiple host-phage systems. The current study aimed to build upon this previous work by examining different temperature conditions at the point of infection to determine the effect on harvest phage titre improvements. Investigations were conducted at different culture scales ranging from 20mL shake flasks to 3L stirred tank bioreactor cultures to investigate process differences when scaling from laboratory bench-scale to initial industrial scale fermentations. Additionally, the kinetics of phage infection were investigated. In small scale cultures, the greatest phage bursts and harvest titres were generated by maintaining cultures under static 28 o C incubation during infection compared to agitation and temperature reduction from 37 o C. Investigating the dynamics around the point of infection will help to inform scalable processes for manufacture of phage for a variety of applications ranging from direct therapeutic application to self-assembling bio-templates for nanostructure synthesis.

Characterization of E coli phage isolated from sewage

Bacteriophage are viruses that infect bacterial cells. as with all viruses, phage are nonliving agents and thus require the use of the host‟s metabolic processes to replicate itself. in this study, the phage of interest are those that infect and lyses E. colt host cells. when phage are released from the ruptured host, distinct zones of clearing (plaques) form. the original E. colt host cells for this experiment came from a sample of raw sewage. in order to obtain the bacteriophage, a procedure of enrichment, isolation, dilution and seeding was followed, the presence of distinct plaques indicated that lytic bacteriophage had been successfully amplified, separated and grown.This study included determination of phage titre, latent period , rise period and the burst size of the phage and effect some of factor on phage titre such as (temperature, ether and chloroform) .for determination ofhage titre used series of dilutions(10-1, 10-2, 10-3, 10-4, 10-4, 10-6, 10-7, 10-8, 10-9) the dilution factor gave the best countable number of plaques is(103). this dilution factor was then used for all other experiments, the latent period , rise period and the burst size of the phage are determined by countable number of plaques and phage titre(titer: plaque-forming unit(p.f.u) during 10,20,30,40,50, and 60 minutes . it was (4.7x105 „ 5.3x105 and 6.0x105)during 1O,20and30minutes respectively in the latent period ,but it was (8.5x105 8.9x10‟ 9.3x105)during 40,50,and 60 minutes respectively in the rise period .then the burst size of the phage is counted by the ratio of the phage titer after rise period to that during the latent period it was(1.67).This study also included effect of temperature on phage titre the statistical analysis was significantly increase P<0.05 in phage titre at the temperature37 C° comparing with phage titre at the temperature 50 C° and phage titre at the temperature 65 C°. effects of ether and chloroform on number of plaques and phage titre during 5,10,15 ,20,25 ,30,35 and 40 minutes it was(0.7x105 , 0.3x105 , 0 , 0 , 0 , 0, 0 and 0) respectively in ether sensitivity, but the phage titre in chloroform sensitivity was completely inactivated by chloroform treatment, the statistical analysis (freedom degree ( 2,21 ) and F value=52.60 was high] significant increase (P<0.05) in phage titre in normal saline comparing with phage titre in ether and chloroform sensitivity

THE EFFECT OF SODIUM CHLORIDE ON PHAGE FORMATION BY STAPHYLOCOCCI AT ELEVATED TEMPERATURES

In experiments with the K strain of Staphylococcus aureus and the K race of bacteriophage suspended in tryptose phosphate broth and maintained at 42°C. it was found that the presence of 1 M NaCl produced certain drastic changes in the relationship between the host cells and the infecting virus: 1. Staphylococci grown at 42°C. in plain broth or in NaCl-broth are "activated," i.e. when growth is stopped by lowering the temperature to 5°C. and phage is added, the activity titre immediately displays a rise of 15- to 16-fold. 2. 1 M NaCl tends to prevent the sorption of phage by cocci and this effect is more pronounced at 42°C. than at 5°C. When the activation test is conducted at 5°C. (the usual temperature) most of the phage is picked up by the cells and the described increase in activity titre follows. If the test takes place at 42°C. there is little sorption and correspondingly little rise in phage titre. 3. Mixtures of staphylococci and phage incubated at 42°C. in NaCl-broth fail to produce phage; the final plaque and activity titres are identical with the initial titres. Here, also, the influence of 1 M NaCl in preventing contact of phage with cocci appears to account for the results. 4. Similar mixtures held at 42°C. in plain broth exhibit a drop of about 60 per cent in activity and plaque titres. The loss of phage may be due to adsorption on dead cells accumulating in the suspension or to the thermolability of the bacterium-phage complex, or to both.