Anti-Acinetobacter baumannii single-chain variable fragments show direct bactericidal activity

Document Type : Original Article


1 Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran

2 Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran

3 Chemical Injuries Research Center, Systems Biology and Poisoning Institute, Baqiyatallah University of Medical Sciences, Tehran, Iran

4 Department of Medical Genetics, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

5 National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran

6 Molecular Medicine Department, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

7 Department of Immunology, Pasteur Institute of Iran, Tehran, Iran



Objective(s): The high resistance rate of Acinetobacter baumannii and the limited number of available antibiotics have prompted a worldwide effort to develop effective antimicrobial agents. Accordingly, identifying single-chain variable fragment antibodies (scFvs), capable of exerting direct antibacterial activity in an immune system-independent manner, may be making immunocompromised patients more susceptible to A. baumannii infections.
Materials and Methods: To isolate bactericidal scFvs targeting A. baumannii, we panned a large human scFv phage display library against whole-cell extensively drug-resistant (XDR) A. baumannii strains grown as biofilm or cultured with human blood or human peripheral blood mononuclear cells plus plasma. The binding of scFv-phages to A. baumannii was assessed by the dot-blot assay. Soluble scFvs, derived from the selected phages, were assessed based on their ability to bind and inhibit the growth of A. baumannii. 
Results: Five phage clones showed the highest reactivity toward A. baumannii. Among five soluble scFvs, derived from positive phage clones, two scFvs, EB211 and EB279, had high expression yields and displayed strong binding to A. baumannii compared with the controls. Moreover, XDR A. baumannii strains treated with positively-charged scFvs, including EB211, EB279, or a cocktail of EB211 and EB279 (200 µg/ml), displayed lower viability (approximately 50%, 78%, and 40% viability, respectively) compared with PBS-treated bacteria.
Conclusion: These results suggest that combining last-resort antibiotics with bactericidal scFvs could provide promising outcomes in immunocompromised individuals with A. baumannii infections.


1. Wang J, Xiong K, Pan Q, He W, Cong Y. Application of TonB-dependent transporters in vaccine development of gram-negative bacteria. Front Cell Infect Microbiol 2020; 10:589115.
2. Isler B, Doi Y, Bonomo RA, Paterson DL. New treatment options against carbapenem-resistant Acinetobacter baumannii infections. Antimicrob Agents Chemother 2019; 63:1110-1118.
3. Harding CM, Hennon SW, Feldman MF. Uncovering the mechanisms of Acinetobacter baumannii virulence. Nat Rev Microbiol 2018; 16:91-102.
4. Zeighami H, Valadkhani F, Shapouri R, Samadi E, Haghi F. Virulence characteristics of multidrug resistant biofilm forming Acinetobacter baumannii isolated from intensive care unit patients. BMC Infect Dis 2019; 19:629.
5. Irani N, Basardeh E, Samiee F, Fateh A, Shooraj F, Rahimi A, et al. The inhibitory effect of the combination of two new peptides on biofilm formation by Acinetobacter baumannii. Microb Pathog 2018; 121:310-317.
6. Bassetti M, Labate L, Russo C, Vena A, Giacobbe DR. Therapeutic options for difficult-to-treat Acinetobacter baumannii infections: A 2020 perspective. Expert Opin Pharmacother 2021; 22:167-177.
7. Wang M, Zhang Y, Zhu J. Anti-Staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice. Appl Microbiol Biotechnol 2016; 100:2153-2162.
8. Soltanmohammadi B, Piri-Gavgani S, Basardeh E, Ghanei M, Azizi M, Khaksar Z, et al. Bactericidal fully human single-chain fragment variable antibodies protect mice against methicillin-resistant Staphylococcus aureus bacteraemia. Clin Transl Immunology 2021; 10:e1302.
9. Richard G, MacKenzie CR, Henry KA, Vinogradov E, Hall JC, Hussack G. Antibody Binding to the O-Specific Antigen of Pseudomonas aeruginosa O6 Inhibits Cell Growth. Antimicrob Agents Chemother 2020; 64:e02168-19.
10. Xie X, McLean MD, Hall JC. Antibody-dependent cell-mediated cytotoxicity-and complement-dependent cytotoxicity-independent bactericidal activity of an IgG against Pseudomonas aeruginosa O6ad. J Immunol 2010; 184:3725-3733.
11. Richard G. Investigating the bactericidal mechanism of anti-LPS antibodies against Pseudomonas aeruginosa serotype O6. Ontario, Canada: University of Guelph 2017; 175.
12. LaRocca TJ, Katona LI, Thanassi DG, Benach JL. Bactericidal action of a complement-independent antibody against relapsing fever Borrelia resides in its variable region. J Immunol 2008; 180:6222-6228.
13. Ahamadi-Fesharaki R, Fateh A, Vaziri F, Solgi G, Siadat SD, Mahboudi F, et al. Single-chain variable fragment-based bispecific antibodies: Hitting two targets with one sophisticated arrow. Mol Ther Oncolytics 2019; 14:38-56.
14. Jacobs AC, Sayood K, Olmsted SB, Blanchard CE, Hinrichs S, Russell D, et al. Characterization of the Acinetobacter baumannii growth phase-dependent and serum responsive transcriptomes. FEMS Immunol Med Microbiol 2012; 64:403-412.
15. Colquhoun JM, Rather PN. Insights into mechanisms of biofilm formation in Acinetobacter baumannii and implications for uropathogenesis. Front Cell Infect Microbiol 2020; 10:253.
16. Mostafa M, Siadat SD, Shahcheraghi F, Vaziri F, Japoni-Nejad A, Yousefi JV, et al. Variability in gene cassette patterns of class 1 and 2 integrons associated with multi drug resistance patterns in Staphylococcus aureus clinical isolates in Tehran-Iran. BMC Microbiol 2015; 15:152.
17. Weinstein MP. Performance standards for antimicrobial susceptibility testing: Clinical and Laboratory Standards Institute; 2019.
18. Kroeger LA, Hovde LB, Mitropoulos IF, Schafer J, Rotschafer JC. Colistin methanesulfonate against multidrug-resistant Acinetobacter baumannii in an in vitro pharmacodynamic model. Antimicrob Agents Chemother 2007; 51:3431-3433.
19. Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindquist E, Schier R, et al. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci USA 1998; 95:6157-6162.
20. Zarei B, Javidan Z, Fatemi E, Jamnani FR, Khatami S, Khalaj V. Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library. DARU J Pharm Sci 2020; 28:221-235.
21. Eisenhardt SU, Schwarz M, Bassler N, Peter K. Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules. Nat Protoc 2007; 2:3063-3073.
22. Brochet X, Lefranc MP, Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res 2008; 36:W503-508.
23. Gasteiger E HC, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. The Proteomics Protocols Handbook 2005; 571-607.
24. Soudeiha MA, Dahdouh EA, Azar E, Sarkis DK, Daoud Z. In vitro evaluation of the colistin-carbapenem combination in clinical isolates of Acinetobacter baumannii using the checkerboard, Etest, and time-kill curve techniques. Front Cell Infect Microbiol 2017; 7:209.
25. Lee C-R, Lee JH, Park M, Park KS, Bae IK, Kim YB, et al. Biology of Acinetobacter baumannii: Pathogenesis, antibiotic resistance mechanisms, and prospective treatment options. Front Cell Infect Microbiol 2017; 7:55.
26. Weber BS, Harding CM, Feldman MF. Pathogenic Acinetobacter: from the cell surface to infinity and beyond. J Bacteriol 2016; 198:880-887.
27. Boll JM, Crofts AA, Peters K, Cattoir V, Vollmer W, Davies BW, et al. A penicillin-binding protein inhibits selection of colistin-resistant, lipooligosaccharide-deficient Acinetobacter baumannii. Proc Natl Acad Sci USA 2016; 113:E6228-E6237.
28. Zahn M, Bhamidimarri SP, Baslé A, Winterhalter M, Van den Berg B. Structural insights into outer membrane permeability of Acinetobacter baumannii. Structure 2016; 24:221-231.
29. Sisakhtpour B, Mirzaei A, Karbasizadeh V, Hosseini N, Shabani M, Moghim S. The characteristic and potential therapeutic effect of isolated multidrug-resistant Acinetobacter baumannii lytic phage. Ann Clin Microbiol Antimicrob 2022; 21:1-11.
30. Vukotic G, Obradovic M, Novovic K, Di Luca M, Jovcic B, Fira D, et al. Characterization, antibiofilm, and depolymerizing activity of two phages active on carbapenem-resistant Acinetobacter baumannii. Front Med 2020; 7:426.
31. Wintachai P, Phaonakrop N, Roytrakul S, Naknaen A, Pomwised R, Voravuthikunchai SP, et al. Enhanced antibacterial effect of a novel Friunavirus phage vWU2001 in combination with colistin against carbapenem-resistant Acinetobacter baumannii. Sci Rep 2022; 12:1-9.
32. Goel VK, Kapil A. Monoclonal antibodies against the iron regulated outer membrane proteins of Acinetobacter baumannii are bactericidal. BMC Microbiol 2001; 1:16-24.
33. Wang-Lin SX, Balthasar JP. Pharmacokinetic and pharmacodynamic considerations for the use of monoclonal antibodies in the treatment of bacterial infections. Antibodies 2018; 7:5.
34. Russo TA, Beanan JM, Olson R, MacDonald U, Cox AD, St. Michael F, et al. The K1 capsular polysaccharide from Acinetobacter baumannii is a potential therapeutic target via passive immunization. Infect Immun 2013; 81:915-922.
35. Nielsen TB, Pantapalangkoor P, Luna BM, Bruhn KW, Yan J, Dekitani K, et al. Monoclonal antibody protects against Acinetobacter baumannii infection by enhancing bacterial clearance and evading sepsis. J Infect Dis 2017; 216:489-501.
36. Wang-Lin SX, Olson R, Beanan JM, MacDonald U, Russo TA, Balthasar JP. Antibody dependent enhancement of Acinetobacter baumannii infection in a mouse pneumonia model. J Pharmacol Exp Ther 2019; 368:475-489.
37. Jahangiri A, Owlia P, Rasooli I, Salimian J, Derakhshanifar E, Aghajani Z, et al. Specific egg yolk immunoglobulin as a promising non-antibiotic biotherapeutic product against Acinetobacter baumannii pneumonia infection. Sci Rep 2021; 11:1-11.
38. Wang-Lin SX, Olson R, Beanan JM, MacDonald U, Balthasar JP, Russo TA. The capsular polysaccharide of Acinetobacter baumannii is an obstacle for therapeutic passive immunization strategies. Infect Immun 2017; 85:00591-17.
39. Takada A, Watanabe S, Okazaki K, Kida H, Kawaoka Y. Infectivity-enhancing antibodies to Ebola virus glycoprotein. J Virol 2001; 75:2324-2330.
40. Lee WS, Wheatley AK, Kent SJ, DeKosky BJ. Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies. Nat Microbiol 2020; 5:1185-1191.
41. LaRocca TJ, Holthausen DJ, Hsieh C, Renken C, Mannella CA, Benach JL. The bactericidal effect of a complement-independent antibody is osmolytic and specific to Borrelia. Proc Natl Acad Sci USA 2009; 106:10752-10757.
42. Monnier P, Vigouroux R, Tassew N. In vivo applications of single chain Fv (variable domain)(scFv) fragments. Antibodies 2013; 2:193-208.
43. Li Z, Krippendorff B-F, Sharma S, Walz AC, Lavé T, Shah DK, editors. Influence of molecular size on tissue distribution of antibody fragments. MAbs 2016; 8:113-119. 
44. da Silva Jr A, Teschke O. Effects of the antimicrobial peptide PGLa on live Escherichia coli. Biochim Biophys Acta Mol Cell Res 2003; 1643:95-103.
45. Smart M, Rajagopal A, Liu WK, Ha BY. Opposing effects of cationic antimicrobial peptides and divalent cations on bacterial lipopolysaccharides. Phys Rev E 2017; 96:042405.
46. Wang Y, Wang L, Yang H, Xiao H, Farooq A, Liu Z, et al. The spider venom peptide lycosin-II has potent antimicrobial activity against clinically isolated bacteria. Toxins 2016; 8:119.
47. De Oliveira DMP, Forde BM, Kidd TJ, Harris PNA, Schembri MA, Beatson SA, et al. Antimicrobial Resistance in ESKAPE Pathogens. Clin Microbiol Rev 2020; 33: e00181-19.
48. Ashurst JV DA. Klebsiella Pneumonia.: In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021; 2021.
49. Medrzycka-Dabrowska W, Lange S, Zorena K, Dabrowski S, Ozga D, Tomaszek L. Carbapenem-resistant Klebsiella pneumoniae infections in ICU COVID-19 patients-A scoping review. J Clin Med 2021; 10:2067.
50. Gellatly SL, Hancock RE. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 2013; 67:159-173.
51. Turner KH, Everett J, Trivedi U, Rumbaugh KP, Whiteley M. Requirements for Pseudomonas aeruginosa acute burn and chronic surgical wound infection. PLoS Genet 2014; 10:e1004518.
52. Babich T, Naucler P, Valik JK, Giske CG, Benito N, Cardona R, et al. Risk factors for mortality among patients with Pseudomonas aeruginosa bacteraemia: A retrospective multicentre study. Int J Antimicrob Agents 2020; 55:105847.
53. Draper LA, Cotter PD, Hill C, Ross RP. The two peptide lantibiotic lacticin 3147 acts synergistically with polymyxin to inhibit Gram negative bacteria. BMC Microbiol 2013; 13:212.
54. El-Sayed Ahmed MAE, Zhong LL, Shen C, Yang Y, Doi Y, Tian GB. Colistin and its role in the Era of antibiotic resistance: An extended review (2000-2019). Emerg Microbes Infect 2020; 9:868-885.
55. Jahangiri A, Neshani A, Mirhosseini SA, Ghazvini K, Zare H, Sedighian H. Synergistic effect of two antimicrobial peptides, Nisin and P10 with conventional antibiotics against extensively drug-resistant Acinetobacter baumannii and colistin-resistant Pseudomonas aeruginosa isolates. Microb Pathog 2021; 150:104700.
56. Thomas VM, Brown RM, Ashcraft DS, Pankey GA. Synergistic effect between nisin and polymyxin B against pandrug-resistant and extensively drug-resistant Acinetobacter baumannii. Int J Antimicrob Agents 2019; 53:663-668.
57. Hancock RE. The bacterial outer membrane as a drug barrier. Trends Microbiol 1997; 5:37-42.
58. Wu X, Li Z, Li X, Tian Y, Fan Y, Yu C, et al. Synergistic effects of antimicrobial peptide DP7 combined with antibiotics against multidrug-resistant bacteria. Drug Des Devel Ther 2017; 11:939-946.
59. van der Linden DS, Short D, Dittmann A, Yu PL. Synergistic effects of ovine-derived cathelicidins and other antimicrobials against Escherichia coli O157:H7 and Staphylococcus aureus 1056 MRSA. Biotechnol Lett 2009; 31:1265-1267.
60. Liu J, Chen F, Wang X, Peng H, Zhang H, Wang KJ. The synergistic effect of mud crab antimicrobial peptides sphistin and Sph12-38 with antibiotics azithromycin and rifampicin enhances bactericidal activity against Pseudomonas aeruginosa. Front Cell Infect Microbiol 2020; 10:572849.