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Phage therapy

Bibliography

Concluding remarks

Background Acinetobacter baumannii is a gram-negative bacterium member of the normal microbiota in humans, which in some situations is able to produce opportunistic infections (1). This bacteria is one of the most problematic nosocomial pathogens in hospitals worldwide due to its high rate of acquisition of resistance to antibiotics (2). Currently, there has been established a set of lines of research to find new treatments in order to provide an alternative to conventional treatments with antibiotics.

Bacteriophage and lysins are treatments with therapeutic potential against multidrug resistant bacteria. Studies have shown different strategies to overcome disadvantages of both methods, a consideration to keep in mind as well as an incentive to apply these treatments. Nevertheless, alternative treatments have not been accepted to use in humans, mainly because of lack of a specific regulatory framework and the requirement of in vivo assays. Studies in phage therapy against A. baumannii has been focused primarily on in vitro assays, whereas phage lysins have been tested in animal model assays with successful results. Future research should be concentrated on clinical trials in animal models and humans in order to show the efficacy and safety of these treatments.

Use of phage therapy to treat bacterial infections is based on the administrationof bacteriophages to the patient in order to eliminate the pathogen that causesthe disease (3, 4).

Phage lysins are enzymes capable of lysing the cell from within during the late phase of the viral lyticcycle, allowing the release of virions (5).

Jordi Corral Sábado - Grau en Microbiologia - Tutor: Jesús Aranda Rodríguez - 30th May 2015

Use of bacteriophages and their lysins as alternative treatments against multidrug resistant Acinetobacter baumannii strains

1. Fournier PE, Richet H. 2006. The epidemiology and control of Acinetobacter baumannii in health care facilities. Clin Infect Dis 42:692-6992. Maragakis LL, Perl TM. 2008. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis 46:1254-12633. Kutter E, De Vos D, Gvasalia G, Alavidze Z, Gogokhia L, Kuhl S, Abedon ST. 2010. Phage therapy in clinical practice: treatment of human infections. Curr Pharm Biotechnol 11:69-864. Drulis-Kawa Z, Majkowska-Skrobek G, Maciejewska B, Delattre AS, Lavigne R. 2012. Learning from bacteriophages - advantages and limitations of phage and phage-encoded protein applications. Curr Protein Pept Sci 13:699-7225. Hermoso JA, García JL, García P. 2007. Taking aim on bacterial pathogens: from phage therapy to enzybiotics. Curr Opin Microbiol 10:461-4726. Yang H, Yu J, Wei H. 2014. Engineered bacteriophage lysins as novel anti-infectives. Front Microbiol 5:5427. Merabishvili M, Vandenheuvel D, Kropinski AM, Mast J, De Vos D, Verbeken G, Noben JP, Lavigne R, Vaneechoutte M, Pirnay JP. 2014. Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii. PLoS One 9:e1048538. Lood R, Winer BY, Pelzek AJ, Diez-Martinez R, Thandar M, Euler CW, Schuch R, Fischetti VA. 2015. Novel Phage Lysin Capable of Killing the Multidrug-Resistant Gram-Negative Bacterium Acinetobacter baumannii in a Mouse Bacteremia Model. Antimicrob Agents Chemother 59:1983-1991

Lysins

1. Viral DNA injected into the cell

2. Assembly of new viruses

3. Lysis of cell

Figure 1: Schematic bacteriophage cycle.

Limitations Strategies to solve

• High specificity• Phage-resistant bacterial• Transduction of genes• Immunogenicity of virions• Toxicity associated to bacterial

lysis• Obtaining phages of non typical

bacteria

• Phage cocktails• Combination of antibiotics and phages• PEGylation of phages• Using lysis-deficient phages• Phage engineering:changing aminoacids

of capsid, combining receptors ofdifferent phages, introduction ofsensitive antibiotic genes and covalentunion of antibiotics

Figure 3: Structure of lysin, chimeolysin (hybrid of two lysins) andartilysin (lysin joined to cationic peptide (CP)). A typical lysin isformed by a catalytic domain (CD), joined to a cell wall bindingdomain (CBD) (6).

N-terminal C-terminal

CP

Frequency of occurrence of phage-

resistant mutant bacterial cells

Host range study of phages

Establishing the A. baumannii phage collection strains

Phage isolation, purification

and propagation

Mouse Acinetobactersepsis model

Expression and purification of

PlyF307

Activity screen of lysins on plates

Generation of phage DNA library

Figure 4: (A) Acibel004. (B) Acibel007. (C) Percentage ofadsorbation (red) and propagation (green) in different strains.(D) Steps in order to determinate phage activity (7).

Phage and bacterial

suspensions

Phage activity

Examples of treatments against multidrug resistant A. baumannii strains

Results of the study• Both phages were able to propagate in approximately half

of the tested A. baumannii strains but 2 strains showedcomplete resistance to both phages.

• The newly isolated phages can serve as potential candidatesfor phage cocktails.

C

D

Results of the study

• Phage lysins from Acinetobacter prophages can be used to efficiently reduce the bacterialburden of the multidrug resistant A. baumannii, both in vitro and in vivo.

Prophage induction and lysin gen insertion in Escherichia

coli DH5α

Figure 5: (A) Steps on the study in order to determinate lysin activity. (B) Percentage of survivalafter infection with A. baumannii within treatment (---) and treated with PlyF307 (___) (8).

A B

Lysin production and mouse sepsis model

Lysin

Chimeolysin

Artilysin

CD

CD

CD

CBD

CBD

CBD

Phage therapy with bacteriophages Acibel004 and Acibel007 (7) Lysin PlyF307 (8)

A B

Limitations

• Immunogenicity of lysins• Toxicity associated to bacterial lysis• Exogenously effective application only in gram-positive

Strategies to solve

• Combination of antibiotics and lysins• Synergic use of two lysins• Phage lysin engineering: chimeolysins and artilysins

Figure 2: Representation of a gram-negativebacterial cell wall (5).