iNtRON Biotechnology has been conducting evaluation tests on the recombinant phage endolysins AP50-31 and LysB4, to validate their bacteriolytic properties against Bacillus genus. As a result, AP50-31 and LysB4 displayed rapid bacteriolytic activity against broad spectrum of Bacillus genus including Bacillus anthracis strains, and the findings were recently published in the world’s top academic journal, Nature’s Scientific Reports.
This research result is a prominent milestone to aid the world to secure an effective defensive measure to prepare for B. anthracis used biowarfare or bioterrorism, and also gives a chance to open new business opportunities in biotechnology based defense industry.
You can find the corresponding article at below.
(https://www.nature.com/search?q=anthrax)
Test Protocol
Experimental group (8 mice) and control group (16 mice) were intranasally challenged with spores of B. anthracis Sterne., then the control group was treated with Buffer (without endolysin), and the experimental group was treated with LysB4 at 6, 24 and 48 hours post-infection. After 3 days of infection, major organs (Lung, liver, kidney, spleen) of each group were extracted to measure bacterial counts in tissues.
in vitro Bacteriolytic Properties
The bacteriolytic activities of AP50-31 and LysB4 were examined using a conventional turbidity reduction assay that measured the time required to reach one-half of the starting absorbance (TOD50) after each phage endolysin was added at a final concentration of 5 μg /mL to a bacterial cell suspension. As a result, AP50-31 and LysB4 displayed broad bacteriolytic spectra within the Bacillus genus and displayed rapid bacteriolytic properties
Susceptibilities of Bacillus strains to AP50-31 and LysB4
Species | Strain | TOD50 (min) | |
AP50-31 | LysB4 | ||
Bacillus cereus | ATCC 4342 | 3.8 ± 0.06 | 2.2 ± 0.06 |
Bacillus circulans | ATCC 21783 | ND | 3.5 ± 0.15 |
Bacillus laevolacticus | ATCC 23492 | ND | 1.4 ± 0.06 |
Bacillus licheniformis | KCOM 1491 | 19.4 ± 0.21 | 2.1 ± 0.06 |
Bacillus megaterium | ATCC 10778 | NS | 2.2± 0.06 |
Bacillus pumilus | KCTC 3713 | ND | 2.6 ± 0.06 |
Bacillus subtilis | RIK 1285 | 15.0 ± 0.06 | 0.5 ± 0.06 |
Bacillus thuringiensis | BGSC 4AA1 BGSC 4AJ1 BGSC 4BA1 BGSC 4CC1 | NS 2.2 ± 0.06 2.8 ± 0.06 2.2 ± 0.00 | 4.2 ± 0.06 NS NS NS |
ND (not determined) : TOD50 could not be determined under this experimental condition, but susceptibility to the corresponding endolysin was confirmed under a concentration of more than 5 μg /mL; NS (not susceptible) : TOD50 could not be determined up to 300 μg /mL of phage endolysin. Data are the mean±standard deviation. The tests were performed three times independently.
Susceptibilities of B. anthracis strains to AP50-31 and LysB4
Species | Strain | TOD50 (min) | |
AP50-31 | LysB4 | ||
B. anthracis ∆ Sterne | None | 9.0 ± 1.73 | 6.3 ± 0.29 |
B. anthracis Sterne | pXO1 | 9.3 ± 0.76 | 8.2 ± 1.04 |
B. anthracis ATCC | pXO1. pXO2 | 16.3 ± 2.02 | 10.2 ± 0.58 |
B. anthracis HYU01 | pXO1. pXO2 | 9.5 ± 1.00 | 7.7 ± 0.29 |
Susceptibilities of B. anthracis strains to AP50-31 and LysB4. Data are the mean±standard deviation. The tests were performed three times independently.
in-vitro Efficacy Test
Challenge with spores of B. anthracis caused physical signs of ill health in the buffer-treated mice that manifested as hunched backs, ruffed fur, piloerection, and ocular discharge. These clinical signs appeared 3-4 days after the bacterial challenge and became aggravated in a time-dependent manner. However, LysB4 treatment markedly improved the clinical signs and no symptoms were observed in the high-dose LysB4-treated group (G3, 100 μg/head).
In addition, LysB4 treatment efficiently rescued the infected mice, and no deaths were observed in the high-dose LysB4-treated group throughout the experimental period. The administration of high-dose LysB4 at 6, 24 and 48 h post-infection provided 100% survival (G3, 100 μg/head), while low-dose LysB4 treatment (G2, 10 μg/head) delayed the onset of death and significantly improved the survival rate. The mean body temperature of the mice decreased corresponding to the onset of clinical signs, and the decrease was more prominent in the buffer-treated group compared with the LysB4-treated group.
Histopathological Analysis
To determine the effect of LysB4 on bacterial clearance, the lungs, livers, spleens, and kidneys were extracted from buffer- or LysB4-treated mice at day 3 post-infection and the total count of bacteria (spores and vegetative cells) in each tissue was determined.
In all of the tissues analyzed, the bacterial count of the LysB4-treated group was significantly lower than that of the buffer-treated group. Furthermore, most of the pathological findings in control group were markedly improved in the organs of the infected and LysB4-treated group, indicating that LysB4 had a protective effect against B. anthracis infection in mice.
Above mentioned endolysins under development for B. anthracis treatment have entirely different mode of action from existing vaccines as they act as a “fundamental” treatment by destroying causative bacteria, and has rapid bacteriolytic activity compared with ciprofloxacin drugs, which makes the endolysins to be considered as the most suitable candidate to treat rapid infection of B. anthracis.
iNtRON Biotechnology will make a constant effort to develop novel and innovative solutions for B. anthracis and Bacillus treatment, and to be a global leader equipped with leading bacteriophage and endolysin technologies.
it is iNtRON.