Formation of therapeutic phage cocktail and endolysin to highly multi-drug resistant Acinetobacter baumannii: in vitro and in vivo study

Document Type : Original Article


1 College of Medicine, AL-Nahrain University, Medical Microbiology Department, Baghdad, Iraq

2 College of Medicine, Baghdad University, Department of Microbiology, Baghdad, Iraq


Objective(s): Phage therapy is a potential alternative treatment for infections caused by Acinetobacter baumannii, a significant nosocomial pathogen, which has evolved resistance to almost all conventional antimicrobial drugs in poor hygiene and conflicts areas such as Iraq.
Materials and Methods: Bacteriophages were isolated to highly resistant isolates of A. baumannii to form therapeutic phage cocktail, and to extract and evaluate native endolysin activity. Bacterial samples were collected in Al-Imamein Al-kadhimein Medical City Hospital. Phages were isolated from different regions in Baghdad city including (soil, sewage, irrigation channels). Phage endolysin was extracted from highly lytic phages that produced halo-like appearance around inhibition zone.
Results: Up to 23 isolates of extensive- and pan- drug resistant (XDR, PDR) A. baumannii were isolated from patients with various infections, and 136 lytic phages specific to A. baumannii were isolated. Each bacterial isolate was sensitive to at least one lytic phage. Accordingly, a phage cocktail was formulated which remarkably minimized bacterial resistance to lysis by phages when compared to individual lytic phages. And, the phage cocktail succeeded in treating and saving life of all bacteremic mice with A. baumannii versus the non-treated group. In addition, the endolysin native activity to A. baumannii was evaluated in this study; endolysin revealed a potent antibacterial activity (> 1 log) reduction of bacterial density in just one hour of endolysin treatment.
Conclusion: The phage therapy assessed in this study showed an ability to efficiently solve the problems of “superbug” bacteria by lysing effectively most XDR, PDR bacteria in vitro and in vivo. And, phage cocktail was shown to be superior over single-phage preparations in treating A. baumannii with much less resistance rate to therapeutic phages. Furthermore, intrinsic activity of native endolysin revealed promising results to tackling superbug pathogens.


Main Subjects

1. Cantas L, Shah S, Cavaco L, Manaia C, Walsh F, Popowska M, et al. A brief multi-disciplinary review on antimicrobial resistance in medicine and its linkage to the global environmental microbiota. Front Microbiol 2013; 4:96-110.          
2. World Health Organization. Antimicrobial resistance: global report on surveillance. World Health Organization 2014.
3. Murray CK, Yun HC, Griffith ME, Thompson B, Crouch HK, Monson LS,  et al. Recovery of multidrug-resistant bacteria from combat personnel evacuated from Iraq and Afghanistan at a single military treatment facility. Mil Med 2009; 174:598-604.
4. Calhoun DA, Jones D, Textor S, Goff DC, Murphy TP, Toto RD, et al. Resistant hypertension: diagnosis, evaluation, and treatment: a scientific statement from the American Heart Association Professional Education Committee of the Council for High Blood Pressure Research. Circulation 2008; 117:510-526.
5. O’Neill J. Tackling drug-resistant infections globally: final report and recommendations. Review of antimicrobial resistance. London: HM Government and Wellcome trust 2016. There is no corresponding record for this reference. 2016.
6. Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM. Phage treatment of human infections. Bacteriophage 2011; 1:66-85.
7. Hendrix RW, Smith MC, Burns RN, Ford ME, Hatfull GF. Evolutionary relationships among diverse bacteriophages and prophages: all the world’sa phage. Proc Natl Acad Sci U S A 1999; 96:2192-2197.
8. Clokie MR, Millard AD, Letarov AV, Heaphy S. Phages in nature. Bacteriophage 2011; 1:31-45.
9. Flores CO, Meyer JR, Valverde S, Farr L, Weitz JS. Statistical structure of host–phage interactions. Proc Natl Acad Sci U S A 2011; 108:E288-E297.
10. Örmälä AM, Jalasvuori M. Phage therapy: should bacterial resistance to phages be a concern, even in the long run?. Bacteriophage 2013; 3:24219-24222.
11. Mattila S, Ruotsalainen P, Jalasvuori M. On-demand isolation of bacteriophages against drug-resistant bacteria for personalized phage therapy. Front Microbiol 2015; 6:1271-1278.
12. Sulakvelidze A, Alavidze Z, Morris JG. Bacteriophage therapy. Antimicrob Agents Chemother 2001; 45:649-659.
13. Chan BK, Abedon ST. Phage therapy pharmacology: phage cocktails. Adv Appl Microbiol 2012; 78:1-23.
14. Falagas ME, Vardakas KZ, Kapaskelis A, Triarides NA, Roussos NS. Tetracyclines for multidrug-resistant Acinetobacter baumannii infections. Int J Antimicrob Agents 2015; 45:455-460.
15. Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel antimicrobials. Future Microbiol 2012; 7:1147-1171.
16. Briers Y, Walmagh M, Lavigne R. Use of bacteriophage endolysin EL188 and outer membrane permeabilizers against Pseudomonas aeruginosa. J Appl Microbiol 2011; 110: 778-785.
17. Briers Y, Walmagh M, Van Puyenbroeck V, Cornelissen A, Cenens W, Aertsen A, et al. Engineered endolysin-based “Artilysins” to combat multidrug-resistant Gram-negative pathogens. MBio 2014; 5:e01379-01314.
18. Merril CR, Scholl D, Adhya SL. The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2003; 2:489-497.
19. WHO (World Health Organization). 2003. Basic laboratory procedures in clinical bacteriology .2nd ed. Geneva, Switzerland. pp: 103-121.
20. Clinical and Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing: Twenty-fourth informational supplement, M100-S24. Clinical and Laboratory Standards Institute (CLSI) 2014; 34.
21. Jassim SA, Abdualamir AS and Abu baker F. Methods for bacteriophage design in "international application published under the patent cooperation (PCT)" (W.I.P. organization, ed.) 2010; Vol.WO2010064044 A1.
22. Biswas B, Adhya S, Washart P, Paul B, Trostel AN, Powell B, et al. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium. Infect immun 2002; 70:204-210.
23. Simmons M, Donovan DM, Siragusa GR, Seal BS. Recombinant expression of two bacteriophage proteins that lyse Clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains. J Agric Food Chem 2010; 58:10330-10337.
24. Mshachal MA, Abdulrahman TR, Khudair MS, Hassan JS. Molecular detection of multidrug resistance Acinetobacter baumannii from different clinical samples. Iraqi J Med Sci 2017; 15:314-323.
25. Al Marjani M, Al-Ammar M, Kadhem E. Occurrence of ESBL and MBL genes in Pseudomonas aeruginosa and Acinetobacter baumannii isolated from Baghdad, Iraq. Int J Cur Res 2013; 5:2482-2486.
26. Qureshi ZA, Hittle LE, O'hara JA, Rivera JI, Syed A, Shields RK, et al. Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance. Clin Infect Dis 2015; 60:1295-1303.
27. Pelletier MR, Casella LG, Jones JW, Adams MD, Zurawski DV, Hazlett KR, et al. Unique structural modifications are present in the LPS from colistin-resistant strains of Acinetobacter baumannii. Antimicrob Agents Chemother 2013; 51:4831-4840.
28. Arroyo LA, Herrera CM, Fernandez L, Hankins JV, Trent MS, Hancock RE. The pmrCAB operon mediates polymyxin resistance in Acinetobacter baumannii ATCC 17978 and clinical isolates through phosphoethanolamine modification of lipid A. Antimicrob Agents Chemother 2011; 55:3743-3751.
29. Jain R, Danziger LH. Multidrug-resistant Acinetobacter infections: an emerging challenge to clinicians. Ann Pharmacother 2004; 38:1449-1459.
30. Rodríguez-Martínez JM, Nordmann P, Ronco E, Poirel L. Extended-spectrum cephalosporinase in Acinetobacter baumannii. Antimicrob Agents Chemother 2010; 54:3484-3488.
31. Bragg R, van der Westhuizen W, Lee JY, Coetsee E, Boucher C. Bacteriophages as potential treatment option for antibiotic resistant bacteria. Adv Exp Med Biol 2014; 807:97-110.
32. Kusradze I, Karumidze N, Rigvava S, Dvalidze T, Katsitadze M, Amiranashvili I, et al. Characterization and testing the efficiency of Acinetobacter baumannii phage vB-GEC_Ab-M-G7 as an antibacterial agent. Front Microbiol 2016; 7:1590-1597.
33. Regeimbal JM, Jacobs AC, Corey BW, Henry MS, Thompson MG, Pavlicek RL, et al. Personalized therapeutic cocktail of wild environmental phages rescues mice from Acinetobacter baumannii wound infections. Antimicrob Agents Chemother 2016; 60:5806-5816.
34. Merabishvili M, Vandenheuvel D, Kropinski AM, Mast J, De Vos D, Verbeken G, et al. Characterization of newly isolated lytic bacteriophages active against Acinetobacter baumannii. PLoS ONE 2014; 9:104853-104864.
35. Łobocka M, Hejnowicz MS, Gagała U, Weber-Dabrowska B, Wegrzyn G, Dadlez M. The first step to bacteriophage therapy—How to choose the correct phage. Phage Therapy: Current Research and Applications; Borysowski, J, Miedzybrodzki, R, Górski, A, Eds. 2014:23-69.
36. Jassim S, Abdulamir A, Abu Bakar F. Phage-based limulus amoebocyte lysate assay for rapid detection of bacteria. WO2011/098820A1. 2011.
37. Kelly D, McAuliffe O, O’Mahony J, Coffey A. Development of a broad-host-range phage cocktail for biocontrol. Bioeng bugs 2011; 2:31-37.
38. Benjamin K Chan, Stephen T Abedon, Catherine Loc-Carrillo Future Microbiol 2013; 8:769-783.
39. Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev 2008; 21:538-582.
40. Lai MJ, Lin NT, Hu A, Soo PC, Chen LK, Chen LH, et al. Antibacterial activity of Acinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both Gram-positive and Gram-negative bacteria. Appl Microbiol Biotechnol 2011; 90:529-539.
41. Briers Y, Volckaert G, Cornelissen A, Lagaert S, Michiels CW, Hertveldt K, et al. Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL. Mol Microbiol 2007; 65:1334-1344.
42. Miroshnikov K, Faizullina N, Sykilinda N, Mesyanzhinov V. Properties of the endolytic transglycosylase encoded by gene 144 of Pseudomonas aeruginosa bacteriophage phiKZ. Biochem (Mosc) 2006; 71:300-305.
43. Paradis-Bleau C, Cloutier I, Lemieux L, Sanschagrin F, Laroche J, Auger M, et al. Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage φKZ gp144 lytic transglycosylase. FEMS Microbiol Lett 2007; 266:201-209.
44. Junn HJ, Youn J, Suh KH, Lee SS. Cloning and expression of Klebsiella phage K11 lysozyme gene. Protein Expr Purif 2005; 42:78-84.
45. Mikoulinskaia GV, Odinokova IV, Zimin AA, Lysanskaya VY, Feofanov SA, Stepnaya OA. Identification and characterization of the metal ion‐dependent l‐alanoyl‐d‐glutamate peptidase encoded by bacteriophage T5. FEBS J 2009; 276:7329-7342.
46. Schmelcher M, Shabarova T, Eugster MR, Eichenseher F, Tchang VS, Banz M, et al. Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent phage endolysin cell wall binding domains. Appl Environ Microbiol 2010; 76:5745-5756.
47. Loessner MJ, Kramer K, Ebel F, Scherer S. C‐terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high‐affinity binding to bacterial cell wall carbohydrates. Mol Microbiol 2002; 44:335-349.
48. Lood R, Winer BY, Pelzek AJ, Diez-Martinez R, Thandar M, Euler CW, et al. Novel phage lysin capable of killing the multidrug-resistant Gram negative bacterium Acinetobacter baumannii in a mouse bacteremia model. Antimicrob Agents Chemother 2015; 59:1983-1991.
49. Hibbing ME, Fuqua C, Parsek MR, Peterson SB. Bacterial competition: surviving and thriving in the microbial jungle. Nat Rev Microbiol 2010; 8:15-25.
50. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, et al. Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clin Infect Dis 2009; 48:1-12.
51. Debarbieux L, Leduc D, Maura D, Morello E, Criscuolo A, Grossi O, et al. Bacteriophages can treat and prevent Pseudomonas aeruginosa lung infections. J Infect Dis 2010; 201:1096-1104.
52. Pires DP, Vilas Boas D, Sillankorva S, Azeredo J. Phage therapy: a step forward in the treatment of Pseudomonas aeruginosa infections. J Virol 2015; 89:7449-7456.
53. McCallin S, Alam Sarker S, Barretto C, Sultana S, Berger B, Huq S, et al. Safety analysis of a Russian phage cocktail: from metagenomics analysis to oral application in healthy human subjects. Virology 2013; 443:187-196.
54. Reyes A, Semenkovich NP, Whiteson K, Rohwer F, Gordon JI. Going viral: next-generation sequencing applied to phage populations in the human gut. Nat Rev Microbiol 2012; 10:607-617.
55. Skurnik M, Pajunen M, Kiljunen S. Biotechnological challenges of phage therapy. Biotechnol Lett 2007; 29:995-1003.
56. Merril CR, Scholl D, Adhya S. Phage Therapy. In Calendar R, editor. The Bacteriophages. New York: Oxford University Press; 2006. P 725-741.