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Clara T.Barcelo, Flor I. Sanchez, Marie Vasse et. al.
http://www.pseudomonas.com/
Presentation by: Juan Barrera
 Antibiotics are the
most effective defense
against bacterial
infections.
 Rapid adaptation of
bacteria to antibiotics
leads to resistance.
 There is mounting
interest in search of
alternatives to
antibiotics. http://www.pulsetoday.co.uk/Pictures/web/w/n/x/Antibiotic_resistance_
test_UTIs_urine_infections_OTC__SPL___single_use_only.jpg
 Gram negative.
 Poses a threat to cystic
fibrosis patients.
 Source of hospital
acquired pneumonia.
 Causes chronic lung
infections.
 Infects wounds and
burns.
http://upload.wikimedia.org/commons/e/ec/Pseudomonas_aeruginosa_cu
lture.JPG
 Provide host
specificity.
 More specific than
antibiotics.
 Phages are able to
inject genetic material
inside host.
 LUZ7 phage has
reduced bacterial
population size of P.
aeruginosa.
http://images.fineartamerica.com/images-medium-large-5/t-
bacteriophages-and-e-coli-eye-of-science.jpg
 P.aeruginosa (PAO1)
treated with LUZ7
bacteriophage.
 Pathogen treated with
streptomycin.
 Treatment with both
streptomycin and
LUZ7.
 Results observed by
counting number of
different growing
colonies (CFU).
Figure 1. Overview of experimental design
Barcelo et. al. (2014)
 Single treatments of phage
and antibiotic reduced
bacterial density over the
first 24 hours.
 Densities rebounded to
control levels by the 70 hour
mark
 Combined treatment caused
significant reduction in
density without regrowth.
 Bacterial density reduction
was stronger when antibiotic
was added with a 12 hour
delay.
 Streptomycin dose had no
significant effect on final
density.
Figure 2. Changes in bacterial density over time
Barcelo et. al. (2014)
 Densities were lower
than expected.
 The combined
treatment was most
significant for the 12h
antibiotic addition.
 Delay of antibiotic
addition resulted in
strongest negative
impact.
Figure 3. Final bacterial densities
Barcelo et. al. (2014)
 Bacteria treated from
streptomycin evolved
higher levels of
resistance.
 Single-strep treatment
resistance reached
maximum levels.
 Resistance values were
lower for populations
where streptomycin
was added to the phage
with a 12h delay.Barcelo et. al. (2014)
Figure 4. Resistance of final bacterial populations.
 Absence of phage
produced a decrease
in resistance.
 Previously exposed
bacteria exhibit
resistance towards
ancestral phage.
Figure 4. Resistance of final bacterial populations.
Barcelo et. al. (2014)
 Resistance against
evolved phage was
lower as compared
with ancestral phage.
 Data suggests
adaptation of phage
to bacteria.
Figure 4. Resistance of final bacterial populations.
Barcelo et. al. (2014)
 Combined treatments
lead to synergistic
suppression of
bacterial density and
less resistance.
 Bacteria most affected
when antibiotic added
at the time when
phages had their
strongest impact.
http://news.bbcimg.co.uk/media/images/66413000/jpg/_66413365_c014722
9-bacteriophages_leaving_host_cell,_artwork-spl.jpg
http://www.rsc.org/images/antibiotic-250_tcm18-96081.jpg
 The study provides evidence of the effectiveness of
antibiotic-phage treatment on P.aeruginosa
pathogen. It also suggests a specific window of
opportunity in which the antibiotic can be added
and have the most effect.
Betts A, Vasse M, Kaltz O, Hochberg ME (2013) Back to the future: evolving bacteriophages to increase their effectiveness
against the pathogen Pseudomonas aeruginosa PAO1. Evol. Appl 6: 1054–1063. doi: 10.1111/eva.12085
Chan BK, Abedon ST, Loc-Carrillo C (2013) Phage cocktails and the future of phage therapy. Future Microbiol 8: 769–783. doi:
10.2217/fmb.13.47
Escobar-Paramo P, Gougat-Barbera C, Hochberg ME (2012) Evolutionary dynamics of separate and combined exposure of
Pseudomonas fluorescens SBW25 to antibiotics and bacteriophage. Evol. Appl 5: 583–592. doi: 10.1111/j.1752-4571.2012.00248.x
Jesus, Blazquez, Oliver Antonio, and Gomez-Gomez Jose-Maria. "Mutation And Evolution Of Antibiotic Resistance: Antibiotics
As Promoters Of Antibiotic Resistance?." Current Drug Targets 3.4 (2002): 345-349. Academic Search Complete. Web. 19 Oct.
2014.
Rodriguez-Rojas A, Rodriguez-Beltran J, Couce A, Blazquez J (2013) Antibiotics and antibiotic resistance: A bitter fight against
evolution. Int. J. Med. Microbiol 303: 293–297. doi: 10.1016/j.ijmm.2013.02.004
http://www.pulsetoday.co.uk/Pictures/web/w/n/x/Antibiotic_resistance_test_UTIs_urine_infections_OTC__SPL___single_
use_only.jpg
http://news.bbcimg.co.uk/media/images/66413000/jpg/_66413365_c0147229-bacteriophages_leaving_host_cell,_artwork-
spl.jpg
http://www.pseudomonas.com/
http://upload.wikimedia.org/commons/e/ec/Pseudomonas_aeruginosa_culture.JPG
http://www.rsc.org/images/antibiotic-250_tcm18-96081.jpg
http://images.fineartamerica.com/images-medium-large-5/t-bacteriophages-and-e-coli-eye-of-science.jpg

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Pseudomonas Aeruginosa Treatment with Phage and Antibiotic

  • 1. Clara T.Barcelo, Flor I. Sanchez, Marie Vasse et. al. http://www.pseudomonas.com/ Presentation by: Juan Barrera
  • 2.  Antibiotics are the most effective defense against bacterial infections.  Rapid adaptation of bacteria to antibiotics leads to resistance.  There is mounting interest in search of alternatives to antibiotics. http://www.pulsetoday.co.uk/Pictures/web/w/n/x/Antibiotic_resistance_ test_UTIs_urine_infections_OTC__SPL___single_use_only.jpg
  • 3.  Gram negative.  Poses a threat to cystic fibrosis patients.  Source of hospital acquired pneumonia.  Causes chronic lung infections.  Infects wounds and burns. http://upload.wikimedia.org/commons/e/ec/Pseudomonas_aeruginosa_cu lture.JPG
  • 4.  Provide host specificity.  More specific than antibiotics.  Phages are able to inject genetic material inside host.  LUZ7 phage has reduced bacterial population size of P. aeruginosa. http://images.fineartamerica.com/images-medium-large-5/t- bacteriophages-and-e-coli-eye-of-science.jpg
  • 5.  P.aeruginosa (PAO1) treated with LUZ7 bacteriophage.  Pathogen treated with streptomycin.  Treatment with both streptomycin and LUZ7.  Results observed by counting number of different growing colonies (CFU). Figure 1. Overview of experimental design Barcelo et. al. (2014)
  • 6.  Single treatments of phage and antibiotic reduced bacterial density over the first 24 hours.  Densities rebounded to control levels by the 70 hour mark  Combined treatment caused significant reduction in density without regrowth.  Bacterial density reduction was stronger when antibiotic was added with a 12 hour delay.  Streptomycin dose had no significant effect on final density. Figure 2. Changes in bacterial density over time Barcelo et. al. (2014)
  • 7.  Densities were lower than expected.  The combined treatment was most significant for the 12h antibiotic addition.  Delay of antibiotic addition resulted in strongest negative impact. Figure 3. Final bacterial densities Barcelo et. al. (2014)
  • 8.  Bacteria treated from streptomycin evolved higher levels of resistance.  Single-strep treatment resistance reached maximum levels.  Resistance values were lower for populations where streptomycin was added to the phage with a 12h delay.Barcelo et. al. (2014) Figure 4. Resistance of final bacterial populations.
  • 9.  Absence of phage produced a decrease in resistance.  Previously exposed bacteria exhibit resistance towards ancestral phage. Figure 4. Resistance of final bacterial populations. Barcelo et. al. (2014)
  • 10.  Resistance against evolved phage was lower as compared with ancestral phage.  Data suggests adaptation of phage to bacteria. Figure 4. Resistance of final bacterial populations. Barcelo et. al. (2014)
  • 11.  Combined treatments lead to synergistic suppression of bacterial density and less resistance.  Bacteria most affected when antibiotic added at the time when phages had their strongest impact. http://news.bbcimg.co.uk/media/images/66413000/jpg/_66413365_c014722 9-bacteriophages_leaving_host_cell,_artwork-spl.jpg http://www.rsc.org/images/antibiotic-250_tcm18-96081.jpg
  • 12.  The study provides evidence of the effectiveness of antibiotic-phage treatment on P.aeruginosa pathogen. It also suggests a specific window of opportunity in which the antibiotic can be added and have the most effect.
  • 13. Betts A, Vasse M, Kaltz O, Hochberg ME (2013) Back to the future: evolving bacteriophages to increase their effectiveness against the pathogen Pseudomonas aeruginosa PAO1. Evol. Appl 6: 1054–1063. doi: 10.1111/eva.12085 Chan BK, Abedon ST, Loc-Carrillo C (2013) Phage cocktails and the future of phage therapy. Future Microbiol 8: 769–783. doi: 10.2217/fmb.13.47 Escobar-Paramo P, Gougat-Barbera C, Hochberg ME (2012) Evolutionary dynamics of separate and combined exposure of Pseudomonas fluorescens SBW25 to antibiotics and bacteriophage. Evol. Appl 5: 583–592. doi: 10.1111/j.1752-4571.2012.00248.x Jesus, Blazquez, Oliver Antonio, and Gomez-Gomez Jose-Maria. "Mutation And Evolution Of Antibiotic Resistance: Antibiotics As Promoters Of Antibiotic Resistance?." Current Drug Targets 3.4 (2002): 345-349. Academic Search Complete. Web. 19 Oct. 2014. Rodriguez-Rojas A, Rodriguez-Beltran J, Couce A, Blazquez J (2013) Antibiotics and antibiotic resistance: A bitter fight against evolution. Int. J. Med. Microbiol 303: 293–297. doi: 10.1016/j.ijmm.2013.02.004 http://www.pulsetoday.co.uk/Pictures/web/w/n/x/Antibiotic_resistance_test_UTIs_urine_infections_OTC__SPL___single_ use_only.jpg http://news.bbcimg.co.uk/media/images/66413000/jpg/_66413365_c0147229-bacteriophages_leaving_host_cell,_artwork- spl.jpg http://www.pseudomonas.com/ http://upload.wikimedia.org/commons/e/ec/Pseudomonas_aeruginosa_culture.JPG http://www.rsc.org/images/antibiotic-250_tcm18-96081.jpg http://images.fineartamerica.com/images-medium-large-5/t-bacteriophages-and-e-coli-eye-of-science.jpg

Editor's Notes

  1. Good afternoon, I am Juan and my presentation today is on a paper entitled “A WINDOW OF OPPORTUNITY TO CONTROL THE BACTERIAL PATHOGEN PSEDOMONAS AERIGINOSA COMBINING ANTIBIOTICS AND PHAGES.” I will first present a bit of background on antibiotic resistance, the pathogen P. aeruginosa, what phage therapy is, then I will go into the experimental design, the results obtained, what they mean, and how this can be useful in the future.
  2. Antibiotics are agent that inhibit or kills microorganisms. Antibiotics are the most effective weapon against bacterial infections. However the evolution of resistance is becoming a major problem in hospitals and in agriculture. The extended therapeutic use, and abuse, of antibiotics produced a selective force leading to the selection and spread of resistant bacteria. Resistance is a result of natural selection, directed by the overuse of antibiotics. Bacteria, that have to face the antibiotic challenge, evolve to acquire resistance and, under this strong selective pressure, only the fittest survive. Evolution occurs by acquisition of new functions by exogenous DNA import, and ii) changes in their own genome allowing the modification of the pre-existing genes by mutation and or genome rearrangements. This method of resistance primarily occurs via horizontal gene transfer.
  3. The bacterium Pseudomonas aeruginosa is found to be resistant to many types of antibiotics and causes high mortality in hospitals. P. aeroginosa strains isolated from hospitals have been found to be resistant to up to 16 different antibiotics. Multidrug-resistant P.aeroginosa infections are becoming increasingly common. One in ten hospital-acquired infections are from Pseudomonas. It is an opportunistic, nonsicomial pathogen of immunocompromised individuals. It is able to infect the pulmonary tract, urinary tract, blood ,wounds, and causes other blood infections. One of the most alarming aspects of P.aeroginosa is its low antibiotic susceptibility. These antibiotic resistance characteristics can be attributed to concerted action of efflux pumps with chromosomally encoded antibiotic resistance genes.
  4. Bacteriophages. What are they??? It’s a virus that infects bacteria. A bacteriophage is able to adhere or attach itself to a specific host where it then proceeds to inject its genetic material. Once inside the host the genetic material is able to replicate itself leading to more phages where they will eventually lize or break through the host. They can then proceed to inject and replicate themselves on surrounding hosts. Due to the fact that bacteriophages are so specific to their host, they can be harmless to the host organism and other beneficial bacteria. Phage therapy is the use of bacteriophages to treat bacterial infections. Phage therapy along with the use of antibiotics has been investigated as a possible effective treatment. This poses an advantage when comparison with antibiotics due to the fact that antibiotics kill both the pathogen bacteria as well as the beneficial bacteria.
  5. The experiment was designed to test Pseudomonas aeruginosa with the lytic bacteriophage LUZ7 and the antibiotic streptomycin with the goal of discovering the effects of single and combined treatments. So the experiment was designed to treat the pathogen with only the antibiotic, and only with the phage, and compare the results with the combined treatment. So P.aerugina pathogen was grown on 24 different well plates containing KB media. Phages were added after 6 hours (To) when bacterial populations were growing exponentially and vulnerable to phage attack. The antibiotic (streptomycin) was added at 3 different time points, one was simultaneously with the addition of the phage, another one with a 12 hour delay and the final one with a 24 hours delay. Also 2 different doses of the antibiotic were added, 100 and 240 microgram/mililiter. The bacterial density was measured at different time points by counting of the number of growing colonies (CFU).
  6. At the end of the experiment (T70) they assessed the surviving populations’ resistance to streptomycin and phage. For the resistance test of the antibiotic they took 1 microliter of the final population and inoculated them on different concentrations of the antibiotic. After 24 hours the bacterial density was measured by means of OD, optical density. Resistance was taken as the (MIC) Minimal Inhibitory concentration. In similar fashion 1microliter of the final bacteria was added to media containing ancestral phage and again Optical density was used to measure bacteria density after 24 hours of growth. The same procedure was done with evolved phage. The evolved phage was taken from bacteria from the experiment perform in the first portion. They added 10% chloroform to the bacteria and the phage containing supernatant was recovered as evolved phage.
  7. There is an increasing interest and attention for alternative treatments against bacterial pathogens due to antibiotic resistance. The combination of antibiotics and phages is a tantalizing possibility. The results obtained from this study show that the combination treatment of antibiotic and phage result in synergistic suppression of bacterial density and less resistance than either treatment alone. Bacteria where most affected when the antibiotic was added when the phages had the most impact on bacterial population density. This suggests an optimal window of opportunity in implementation of the combined treatment to restrain most effectively the pathogen. The study also shows evidence of evolution of phage resistance but also evolutionary change in phage infectivity. This indicates that phages can evolve or co-evolve with the bacteria and potentially limit resistance during treatment. The combined phage-antibiotic therapy is expected to have an advantage over antibiotic cocktails because antibiotics and phages are fundamentally different..