Antibacterial activity evaluation of a novel K3-specific phage against <i>Acinetobacter baumannii</i> and evidence for receptor-binding domain transfer across morphologies

Xiangkuan Zheng; Meihan Liu; Pei Li; Sixiang Xu; Long Chen; Guoxin Xu; Xiaoxiao Pang; Hong Du; Yishan zheng; Xiang Huo; Zhongming Tan; Juan Li; Zhirong Li; Wei Zhang

doi:10.1016/j.virs.2024.08.002

October 2024

Citation: Xiangkuan Zheng, Meihan Liu, Pei Li, Sixiang Xu, Long Chen, Guoxin Xu, Xiaoxiao Pang, Hong Du, Yishan zheng, Xiang Huo, Zhongming Tan, Juan Li, Zhirong Li, Wei Zhang. Antibacterial activity evaluation of a novel K3-specific phage against Acinetobacter baumannii and evidence for receptor-binding domain transfer across morphologies .VIROLOGICA SINICA, 2024, 39(5) : 767-781. http://dx.doi.org/10.1016/j.virs.2024.08.002

Antibacterial activity evaluation of a novel K3-specific phage against Acinetobacter baumannii and evidence for receptor-binding domain transfer across morphologies

Xiangkuan Zheng ^a ,
Meihan Liu ^a ,
Pei Li ^a ,
Sixiang Xu ^a ,
Long Chen ^b ,
Guoxin Xu ^b ,
Xiaoxiao Pang ^b ,
Hong Du ^c ,
Yishan zheng ^d ,
Xiang Huo ^e ,
Zhongming Tan ^e ,
Juan Li ^f ,
Zhirong Li ^g ,
Wei Zhang ^{a
,,}

cstr: 32224.14.j.virs.2024.08.002

a. College of Veterinary Medicine, Nanjing Agricultural University, Key Lab of Animal Bacteriology, Ministry of Agriculture and Rural Affairs, Nanjing, 210095, China;
b. Department of Clinical Laboratory, Zhangjiagang Hospital Affiliated to Soochow University, Zhangjiagang, 215600, China;
c. Department of Clinical Laboratory, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, China;
d. Intensive Care Unit, The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003, China;
e. Jiangsu Provincial Center for Disease Prevention and Control, Nanjing, 210009, China;
f. National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Disease, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, 102206, China;
g. Provincial Center for Clinical Laboratories, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, China

Corresponding author: Wei Zhang, vszw@njau.edu.cn
Received Date: 09 April 2024
Accepted Date: 31 July 2024
Available online: 03 August 2024

Abstract

Acinetobacter baumannii (A. baumannii) poses a serious public health challenge due to its notorious antimicrobial resistance, particularly carbapenem-resistant A. baumannii (CRAB). In this study, we isolated a virulent phage, named P1068, from medical wastewater capable of lysing CRAB, primarily targeting the K3 capsule type. Basic characterization showed that P1068 infected the A. baumannii ZWAb014 with an optimal MOI of 1, experienced a latent period of 10 min and maintained stability over a temperature range of 4-37 °C and pH range of 3-10. Phylogenetic and average nucleotide identity analyses indicate that P1068 can be classified as a novel species within the genus Obolenskvirus of the Caudoviricetes class as per the most recent virus classification released by the International Committee on Taxonomy of Viruses (ICTV). Additionally, according to classical morphological classification, P1068 is identified as a T4-like phage (Myoviridae). Interestingly, we found that the tail fiber protein (TFP) of P1068 shares 74% coverage and 88.99% identity with the TFP of a T7-like phage (Podoviridae), AbKT21phiIII (NC_048142.1). This finding suggests that the TFP gene of phages may undergo horizontal transfer across different genera and morphologies. In vitro antimicrobial assays showed that P1068 exhibited antimicrobial activity against A. baumannii in both biofilm and planktonic states. In mouse models of intraperitoneal infection, P1068 phage protected mice from A. baumannii infection and significantly reduced bacterial loads in various tissues such as the brain, blood, lung, spleen, and liver compared to controls. In conclusion, this study demonstrates that phage P1068 might be a potential candidate for the treatment of carbapenem-resistant and biofilm-forming A. baumannii infections, and expands the understanding of horizontal transfer of phage TFP genes.
- Phage
- , A. baumannii
- , Carbapenem-resistant
- , Capsule type
- , Tail fiber protein
- , Antibacterial activity

Electronic Supplementary Material

10.1016j.virs.2024.08.002-ESM.docx
References
1. Alcock BP, Huynh W, Chalil R, Smith KW, Raphenya AR, Wlodarski MA, Edalatmand A, Petkau A, Syed SA, Tsang KK, Baker SJC, Dave M, McCarthy MC, Mukiri KM, Nasir JA, Golbon B, Imtiaz H, Jiang X, Kaur K, Kwong M, Liang ZC, Niu KC, Shan P, Yang JYJ, Gray KL, Hoad GR, Jia B, Bhando T, Carfrae LA, Farha MA, French S, Gordzevich R, Rachwalski K, Tu MM, Bordeleau E, Dooley D, Griffiths E, Zubyk HL, Brown ED, Maguire F, Beiko RG, Hsiao WWL, Brinkman FSL, Van Domselaar G, McArthur AG, 2022. CARD 2023: expanded curation, support for machine learning, and resistome prediction at the comprehensive antibiotic resistance database. Nucleic Acids Res. 51: D690-D699.
2. Ando H, Lemire S, Pires DP, Lu TK, 2015. Engineering modular viral scaffolds for targeted bacterial population editing. Cell. Syst. 1:187-196.
3. Bartal C, Rolston KVI, Nesher L, 2022. Carbapenem-resistant Acinetobacter baumannii: colonization, infection and current treatment options. Infect. Dis. Ther. 11:683-694.
4. Bouras G, Nepal R, Houtak G, Psaltis AJ, Wormald P-J, Vreugde S, 2022. Pharokka: a fast scalable bacteriophage annotation tool. Bioinformatics. 39:btac776.
5. Cahill SM, Hall RM, Kenyon JJ, 2022. An update to the database for Acinetobacter baumannii capsular polysaccharide locus typing extends the extensive and diverse repertoire of genes found at and outside the K locus. Microb. Genom. 8, mgen000878.
6. Cai R., Li D., Lin W., Qin W., Pan L., Wang F., Qian M., Liu W., Zhou Q., Zhou C., Tong Y., 2022. Genome sequence of the novel freshwater Microcystis cyanophage Mwe-Yong1112-1. Arch. Virol. 167:2371-2376.
7. Cano EJ, Caflisch KM, Bollyky PL, Van Belleghem JD, Patel R, Fackler J, Brownstein MJ, Horne B, Biswas B, Henry M, Malagon F, Lewallen DG, Suh GA, 2021. Phage therapy for limb-threatening prosthetic knee Klebsiella pneumoniae infection: case report and in vitro characterization of anti-biofilm activity. Clin. Infect. Dis. 73:e144-e151.
8. Chen M., Zhang L., Abdelgader SA., Yu L., Xu J., Yao H., Lu C., Zhang W., 2017. Alterations in gp37 expand the host range of a T4-like phage. Appl. Environ. Microbiol. 83:e01576-17.
9. Dedrick RM, Smith BE, Cristinziano M, Freeman KG, Jacobs-Sera D, Belessis Y, Whitney Brown A, Cohen KA, Davidson RM, van Duin D, Gainey A, Garcia CB, Robert George CR, Haidar G, Ip W, Iredell J, Khatami A, Little JS, Malmivaara K, McMullan BJ, Michalik DE, Moscatelli A, Nick JA, Tupayachi Ortiz MG, Polenakovik HM, Robinson PD, Skurnik M, Solomon DA, Soothill J, Spencer H, Wark P, Worth A, Schooley RT, Benson CA, Hatfull GF, 2022. Phage therapy of mycobacterium infections: compassionate use of phages in 20 patients with drug-resistant mycobacterial disease. Clin. Infect. Dis. Off Publ. Infect. Dis. Soc. Am. 76:103-112.
10. Domingues R, Barbosa A, Santos SB, Pires DP, Save J, Resch G, Azeredo J, Oliveira H, 2021. Unpuzzling friunavirus-host interactions one piece at a time: phage recognizes Acinetobacter pittii via a new K38 capsule depolymerase. Antibiotics. 10:1304.
11. Ghaffoori Kanaan MH, Al-Shadeedi SMJ, Al-Massody AJ, Ghasemian A, 2020. Drug resistance and virulence traits of Acinetobacter baumannii from Turkey and chicken raw meat. Comp. Immunol. Microbiol. Infect. Dis. 70:101451.
12. Gilchrist CLM, Chooi Y-H, 2021. Clinker & clustermap.js: automatic generation of gene cluster comparison figures. Bioinformatics. 37:2473-2475.
13. Gong X., Zhao Q., Wu Y., Zhou H., Ding S., Zhu K., 2022. Mucoid Acinetobacter baumannii enhances anti-phagocytosis through reducing C3b deposition. Front. Med. 9, 879361.
14. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S, 2018. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat. Commun. 9:5114.
15. Jault P, Leclerc T, Jennes S, Pirnay JP, Que Y-A, Resch G, Rousseau AF, Ravat F, Carsin H, Le Floch R, Schaal JV, Soler C, Fevre C, Arnaud I, Bretaudeau L, Gabard J, 2019. Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1/2 trial. Lancet Infect. Dis. 19:35-45.
16. Kon H, Schwartz D, Temkin E, Carmeli Y, Lellouche J, 2020. Rapid identification of capsulated Acinetobacter baumannii using a density-dependent gradient test. BMC Microbiol. 20:285.
17. Latka A, Lemire S, Grimon D, Dams D, Maciejewska B, Lu T, Drulis-Kawa Z, Briers Y, 2021. Engineering the modular receptor-binding proteins of Klebsiella phages switches their capsule serotype specificity. mBio. 12:e00455-21.
18. Li M., Shi D., Li Y., Xiao Y., Chen M., Chen L., Du H., Zhang W., 2020. Recombination of T4-like phages and its activity against pathogenic Escherichia coli in planktonic and biofilm forms. Virol. Sin. 35:651-661.
19. Linz B, Mukhtar N, Shabbir MZ, Rivera I, Ivanov YV, Tahir Z, Yaqub T, Harvill ET, 2018. Virulent epidemic pneumonia in sheep caused by the human pathogen Acinetobacter baumannii. Front. Microbiol. 9, 2616.
20. Liu B., Zheng D., Zhou S., Chen L., Yang J., 2021. VFDB 2022: a general classification scheme for bacterial virulence factors. Nucleic Acids Res. 50:D912-D917.
21. Liu D., Liu Z-S., Hu P., Hui Q., Fu B-Q., Lu S-Y., Li Y-S., Zou D-Y., Li Z-H., Yan D-M., Ding Y-X., Zhang Y-Y., Zhou Y., Liu N-N., Ren H-L., 2016. Characterization of a highly virulent and antimicrobial-resistant Acinetobacter baumannii strain isolated from diseased chicks in China. Microbiol. Immunol. 60:533-539.
22. Mahichi F, Synnott AJ, Yamamichi K, Osada T, Tanji Y, 2009. Site-specific recombination of T2 phage using IP008 long tail fiber genes provides a targeted method for expanding host range while retaining lytic activity. FEMS Microbiol. Lett. 295:211-217.
23. McConnell MJ, Actis L, Pachon J, 2013. Acinetobacter baumannii: human infections, factors contributing to pathogenesis and animal models. FEMS Microbiol. Rev. 37:130-155.
24. Mea HJ, Yong PVC, Wong EH, 2021. An overview of Acinetobacter baumannii pathogenesis: motility, adherence and biofilm formation. Microbiol. Res. 247:126722.
25. Neto S, Vieira A, Oliveira H, Espina B, 2023. Assessing Acinetobacter baumannii virulence and treatment with a bacteriophage using zebrafish embryos. FASEB. J. 37:e23013.
26. Oliveira H, Costa AR, Ferreira A, Konstantinides N, Santos SB, Boon M, Noben J-P, Lavigne R, Azeredo J, 2019. Functional analysis and antivirulence properties of a new depolymerase from a myovirus that infects Acinetobacter baumannii capsule K45. J. Virol. 93:e01163-18.
27. Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, Fookes M, Falush D, Keane JA, Parkhill J, 2015. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics. 31:3691-3693.
28. Pailhories H, Kempf M, Belmonte O, Joly-Guillou M-L, Eveillard M, 2016. First case of OXA-24-producing Acinetobacter baumannii in cattle from Reunion Island, France. Int. J. Antimicrob. Agents 48:763-764.
29. Pakharukova N, Tuittila M, Paavilainen S, Malmi H, Parilova O, Teneberg S, Knight SD, Zavialov AV, 2018. Structural basis for Acinetobacter baumannii biofilm formation. Proc. Natl. Acad. Sci. U. S. A. 115:5558-5563.
30. Popova AV, Lavysh DG, Klimuk EI, Edelstein MV, Bogun AG, Shneider MM, Goncharov AE, Leonov SV, Severinov KV, 2017. Novel Fri1-like Viruses infecting Acinetobacter baumannii-vB_AbaP_AS11 and vB_AbaP_AS12-characterization, comparative genomic analysis, and host-recognition strategy. Viruses. 9:188.
31. Popova AV, Shneider MM, Arbatsky NP, Kasimova AA, Senchenkova SN, Shashkov AS, Dmitrenok AS, Chizhov AO, Mikhailova YV, Shagin DA, Sokolova OS, Timoshina OY, Kozlov RS, Miroshnikov KA, Knirel YA, 2021. Specific interaction of novel Friunavirus phages encoding tailspike depolymerases with corresponding Acinetobacter baumannii capsular types. J. Virol. 95:e01714-e01720.
32. Ramirez MS, Bonomo RA, Tolmasky ME, 2020. Carbapenemases: transforming Acinetobacter baumannii into a yet more dangerous menace. Biomolecules. 10:720.
33. Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, Barr JJ, Reed SL, Rohwer F, Benler S, Segall AM, Taplitz R, Smith DM, Kerr K, Kumaraswamy M, Nizet V, Lin L, McCauley MD, Strathdee SA, Benson CA, Pope RK, Leroux BM, Picel AC, Mateczun AJ, Cilwa KE, Regeimbal JM, Estrella LA, Wolfe DM, Henry MS, Quinones J, Salka S, Bishop-Lilly KA, Young R, Hamilton T, 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob. Agents Chemother. 61:e00954-17.
34. Shan W., Zhang H., Kan J., Yin M., Zhang J., Wan L., Chang R., Li M., 2021. Acquired mucoid phenotype of Acinetobacter baumannii: Impact for the molecular characteristics and virulence. Microbiol. Res. 246:126702.
35. Shields RK, Paterson DL, Tamma PD, 2023. Navigating available treatment options for carbapenem-resistant Acinetobacter baumannii-calcoaceticus complex infections. Clin. Infect. Dis. 76:S179-S193.
36. Singh JK, Adams FG, Brown MH, 2019. Diversity and function of capsular polysaccharide in Acinetobacter baumannii. Front. Microbiol. 9:3301.
37. Tettelin H, Masignani V, Cieslewicz MJ, Donati C, Medini D, Ward NL, Angiuoli SV, Crabtree J, Jones AL, Durkin AS, DeBoy RT, Davidsen TM, Mora M, Scarselli M, Margarit y Ros I, Peterson JD, Hauser CR, Sundaram JP, Nelson WC, Madupu R, Brinkac LM, Dodson RJ, Rosovitz MJ, Sullivan SA, Daugherty SC, Haft DH, Selengut J, Gwinn ML, Zhou L, Zafar N, Khouri H, Radune D, Dimitrov G, Watkins K, O'Connor KJB, Smith S, Utterback TR, White O, Rubens CE, Grandi G, Madoff LC, Kasper DL, Telford JL, Wessels MR, Rappuoli R, Fraser CM, 2005. Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial “pan-genome” Proc. Natl. Acad. Sci. U. S. A. 102:13950-13955.
38. Wick RR, Judd LM, Gorrie CL, Holt KE, 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput. Biol. 13:e1005595.
39. Wong D., Nielsen TB, Bonomo RA, Pantapalangkoor P, Luna B, Spellberg B, 2016. Clinical and pathophysiological overview of acinetobacter infections: a century of challenges. Clin. Microbiol. Rev. 30:409-447.
40. Wyres KL, Cahill SM, Holt KE, Hall RM, Kenyon JJ, 2020. Identification of Acinetobacter baumannii loci for capsular polysaccharide (KL) and lipooligosaccharide outer core (OCL) synthesis in genome assemblies using curated reference databases compatible with Kaptive. Microb. Genom. 6:e000339.
41. Yoichi M, Abe M, Miyanaga K, Unno H, Tanji Y, 2005. Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157:H7. J. Biotechnol. 115:101-107.
Proportional views