Name: Sarah

Title: Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections
Authors: Laurent Debarbieux, Dominique Leduc, Damien Maura, et al.
Journal: Journal of Infectious Diseases
Date: May 2014

As bacteria are becoming more resistant to antibiotics, this study experiments phage therapy as a
substitute for antibiotics. The purpose of this study is to assess and present a possible cure and prevention by the
method of infecting bacteria with a virus called bacteriophage, which only contaminates and reproduces within
bacteria. The main conclusions of this study are the prevention and cure of bacterial infections that are caused by
the Pseudomonas aeruginosa bacterium. The authors infected mice with P. aeruginosa strain (PAK lumi) and
they died after two days post infection. They concluded that if the test subject is infected and given treatment 2
hours after being infected, with a 10:1 ratio of phage-to-bacterium dosage, the survival rate would be 100%. The
subjects were treated with the bacteriophage treatment after 4 hours of infection, and the survival rate declined
down to 75% and continued to decrease down to a staggering 25% survival rate one the treatment was given 6
hours after infection. With a 1:1 phage-to-bacterium dosage, the survival rate was 80%, good but not good
enough. Therefore, the authors established that the best dose dependency that increases survival rate is a 10:1
ratio. Treatment with bacteriophage showed decreased inflammation in contrast to test subjects left untreated and
only given PBS which did show inflammation. In the process of this experiment, the authors figured out that
bacteriophages could potentially be used as a prevention against bacterial infections in the immunocompromised.
Bacteriophage treatment was given 24 hours before infection and resulted in 100% effectiveness and survival rate.
The authors used bioluminescence to monitor the treatments and preventative treatments to gather statistics and
look for inflammation responses during the experiment. I feel that the study was very good and explained the
procedures and the methods/materials that were used in very clear terms. However I was interested in seeing how
the experiment would have resulted with a bigger sample size. The sample size the authors used in both
experiments, with the PBS (12) and the Phage (12) seemed to be too small of a sample to rely on. Although the
study showed differences between each group, if the experiments are proved to be precise, I believe a higher
sample size for both groups would be more dependable and would have statistics with higher precision. The
authors chose rats that were 8-week-old Balb/C males as a control for their sample to obtain more accurate results.
There was no blinding done in the study from what I could find in the analysis. If blinding methods were used, it
would need to be listed under the materials/methods section, as it would have been a type of method used in an
experiment. Bacteriophages were used here as a preventative against bacterial infection because they only target
bacteria and it takes a longer time for the body to get rid of bacteriophages than initially though. Results showed
that if the mice that were given the bacteriophage 24 hours before they were infected by the bacteria; they will be
protected against infection and will not catch the infection. The mice were infected 24 hours after being given the
PAK-P1 strain and showed 100% survival rate, all four mice survived for 16 days until the experiment ended.
When the mice were pretreated with PBS and infected 24 hours later, all four mice in this group died within 48
hours. These numbers could potentially lead to the possibility of using bacteriophages as a prevention method. In
Figure 1A, this shows the correlation between the time and dose amounts given to survival rates. The authors
show that using a 10:1 or 1:1 ratios of bacteriophages to the PAK-P1 bacteria strain, result in an increased
survival rate compared to lower doses. Using a 10:1 dose-dependency ratio yielded a higher survival rate which
was 100%, than using a 1:1 ratio which yielded an 80% survival rate. I agree with these conclusions because more
the more bacteriophages given in comparison to bacteria, the higher the survival rates. Although the 10:1 ratio
showed to be a more effective dose, the sample size was too small to consider it accurate seeing as the 1:1 ratio
was a very close tie. The purpose of establishing the survival curve was to find a correct dose-dependency that
eliminates bacteria and yields high survival rates. The authors figured out, through the survival curve, that using a
10:1 bacteriophage-to-bacteria ratio resulted in all of the test subjects surviving (100%). The conclusions that the
authors draw from figures 1B and 1C is that subjects treated with a dose of 10:1 ratio bacteriophages (Phage),
were rid of bacterial infection and had 100% survival rate, and this was displayed through bioluminescence in the
mice and compared to the bioluminescence in the mice that were given PBS. In my opinion it is hard to gather
precise evidence without a larger sample size but I do agree with these conclusions, Figure 1B shows the light
emitted wasnt too different from 2 hours and 4 hours, they seemed to look the same in both the phage-treated
subjects and the non-phage treated subjects. Once it reached hour 6, the difference varied greatly and the mice
treated with PBS emitted a lot of light and it was easy to see that the light emitted in the mice treated with
bacteriophages was significantly less. It was necessary to include Figure 1C because it visually describes the
amount of light in quantifiable amounts. In order to have a good description of Figure 1B, Figure 1C was included
to precisely show the numbers of the amount of light given off in the mice in the PBS treatment and the Phage
treatment, as one cannot determine them only by looking at an image like Figure 1B. The conclusions that the
authors draw from figure 2 is the amount of IL-6 and tumor necrosis factor were both indicators that were
increased in the data of the infected mice IL-6 increased to ~6500 pg/ml according to the graph and TNF-alpha
increased to ~1500 pg/ml. Both of these genes give off a response to inflammation. Also the authors figured out
that bacteriophages did not appear to cause inflammation when administered on their own. I agree with the
outcome of the conclusions but feel that the sample size couldnt produce good enough evidence, as the amount of
mice being tested (4) were too few to get a dependable outcome. The purpose of using PBS was to regulate the
pH in the mice being tested. A reaction could occur during the experiment that could cause a low pH response
(acidic) or high pH response (basic) and could affect the experiment. PBS helps to keep the pH levels balanced
and the same so they could be sure pH levels wouldnt have an effect on the experiment, which is also known as a
negative control. IL-6 and Tumor necrosis factor alpha, both have similar functions that trigger inflammation
signals if an infection has occurred. They help recognize and remove unknown substances that enter the body and
is the main response system for an animals immune system. The authors were measuring these two because they
can help get rid of an infection and play a big part in it. The conclusions that the authors draw from Figures 3A
and 3B is that the effectiveness and the rate at which the test subjects survived depended on the timing that the
bacteriophage treatment was administered to the test subject. This figure shows that if a treatment with a 10:1
phage-to-bacterium dose was given within 2 hours of infection, the survival rate was 100%. When the test
subjects were treated 4 hours after infection, there was only 75% survival rate and treatment after 6 hours the
survival rate was 25%. This figure also showed that the mice which were only given phosphate buffered solution,
had a 100% mortality rate and died out within 52 hours. I agree with these conclusions because it showed that the
faster the infected mice were treated, the higher the survival rate was. Although, I think it wouldve been
interesting to see treatments being given in more diverse time frames with more test subjects (e.g. every hour).
Based on this data, I dont think I can conclude that bacteriophages could be used effectively to prevent
Pseudomonas aeruginosa in other organisms. Although when the treatment was administered 24h before
infection, there was 100% survival rate, in my opinion I dont think it would work very well in human beings
because nobody knows the exact time they will get the infection. By the time they notice the symptoms, it might
be too late, the body would eventually eliminate the bacteriophages and the chances of success for treating this
infection would be reduced, so it cant be used as a long term preventative treatment. In Figure 4, it is showing the
effectiveness of the bacteriophage given as a preventative treatment 24 hours before infection could possibly be a
completely effective form of pretreatment and could potentially be used as a vaccine. They support their
conclusions by demonstrating a 100% survival rate. In my opinion from the data shown in this figure, I would
agree with the conclusions, but its hard to prove that this could actually be used to prevent infection without a
higher sample number of test subjects. The conclusion that the authors draw from figure 5 is the PAK-P1 is
highly efficient at removing bacteria, but also the PAK-P1 bacteriophage is very lethal. Considering that the
bacteriophage eliminated bacteria from the body in many trials and time intervals. For example after the
bacteriophage treatment was administered two hours after infection of bacteria, the survival rate was 100% and
when bacteriophage was given as a pretreatment 24 hours before infection, again 100% of the mice survived.
These statistics lead me to agree with the authors conclusion from Figure 5. BLASTp, which is short for Basic
Local Alignment Search Tool for Proteins, is a tool that arithmetically uses a system, which figures out amino
acid and nucleotide sequences in an organisms DNA. It was used in this experiment to compare resemblances
between the capsid proteins and nucleotide sequences (determined by BLASTP) of PAK-P1 and P. Aeruginosa
bacteriophages. I think bacteriophage treatment would be useful in hospital environments with patients who are
immunocompromised and have cancer or cystic fibrosis, etc. These patients are more likely to get infections such
as P. aeruginosa in the lungs because they mainly target patients with weak immune systems. Healthy people
wouldnt really benefit from bacteriophage treatment. The authors also figured out that the PAK-P1 bacteriophage
had more effectiveness in treating minor infections that are secluded but not extensive sustained infections. They
realized they would have to gather particular bacteriophages and improve the study. Yes I do agree with the
authors because in hospitals, patients with weak immune systems who get infected by bacteria like this, tend to
not survive, therefore bacteriophages could potentially reduce mortality rates in hospitals among the
immunocompromised. The downside to their experiment is the small amount of test subjects they sampled, so the
data wasnt dependable enough to say that bacteriophages could prevent infection 100% up to an entire 24 hours,
but it is a good start.