Genomic and evolutionary perspectives on bacteriophage ΦAfa-NA1 targeting MDR Gram-negative bacteria in diabetic ulcer
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Abstract. Narulita E, Utami DAW, Izzaturrohmah L, Arrachmi HI, Febrianti RA, Ludfillah RZ, Widianto AH, Addy HS, Utami BR, Kuswati, Fikri K. 2025. Genomic and evolutionary perspectives on bacteriophage ?Afa-NA1 targeting MDR Gram-negative bacteria in diabetic ulcer. Biodiversitas 26: 6014-6024. The rise of Multidrug-Resistant (MDR) bacterial infections poses a significant challenge in clinical settings, particularly for diabetic ulcer patients who are highly susceptible to persistent infections. This study reports the isolation, characterization, and genomic analysis of a novel lytic bacteriophage, ?Afa-NA1, specifically targeting MDR Gram-negative bacteria associated with diabetic ulcers in Indonesia. The bacteriophage was isolated from wound dressings and tested against four clinical isolates: Alcaligenes faecalis T17, Pseudomonas sp. FP1911, and the Gram-positive control Streptomyces violaceoruber S21. ?Afa-NA1 exhibited strong lytic activity against three Gram-negative isolates but none against the Gram-positive control, confirming host specificity. ?Afa-NA1 produced clear plaques with an average diameter of 3.0±0.5 mm on Alcaligenes faecalis T17, indicative of its potent lytic activity. Stability tests showed the phage retained >90% infective titer after 24 hours at -20°C and 4°C, and exhibited robust stability across a wide pH range (pH 6 to 11). One-step growth analysis revealed a short latent period of 21.7±8.1 minutes and a burst size of 27.7±1.5 PFU per infected cell. Genomic analysis showed ?Afa-NA1 possesses a 78,105 bp double-stranded DNA genome with 45.8% G+C content encoding 83 predicted open reading frames. Comparative genomics and phylogenetic reconstruction confirmed its placement among T7-like Podoviruses, consistent with its obligate lytic lifecycle and genomic architecture. However, the potential for therapeutic application remains preliminary, as host range testing was conducted on a limited panel of single clinical isolates, and all assays were performed in vitro.
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References
Abedon ST, Danis-Wlodarczyk KM, Wozniak DJ. 2021. Phage cocktail development for bacteriophage therapy: Toward improving the spectrum of activity breadth and depth. Pharmaceuticals 14 (10): 1019. DOI: 10.3390/ph14101019.
Atlaw A, Kebede HB, Abdela AA, Woldeamanuel Y. 2022. Bacterial isolates from diabetic foot ulcers and their antimicrobial resistance profile from selected hospitals in Addis Ababa, Ethiopia. Front Endocrinol 13: 987487. DOI: 10.3389/fendo.2022.987487.
Canfield GS, Chatterjee A, Espinosa J, Mangalea MR, Sheriff EK, Keidan M, McBride SW, McCollister BD, Hang HC, Duerkop BA. 2021. Lytic bacteriophages facilitate antibiotic sensitization of Enterococcus faecium. Antimicrob Agents Chemother 65 (5): e00143-21. DOI: 10.1128/aac.00143-21.
Choi HJ, Kim M. 2021. Improved bactericidal efficacy and thermostability of Staphylococcus aureus-specific bacteriophage SA3821 by repeated sodium pyrophosphate challenges. Sci Rep 11 (1): 22951. DOI: 10.1038/s41598-021-02446-1.
Cisek AA, D?browska I, Gregorczyk KP, Wy?ewski Z. 2017. Phage therapy in bacterial infections treatment: One hundred years after the discovery of bacteriophages. Curr Microbiol 74 (2): 277-283. DOI: 10.1007/s00284-016-1166-x.
Das IJ, Bal T. 2024. pH factors in chronic wounds and pH-responsive polysaccharide-based hydrogel dressings. Intl J Biol Macromol 279 (Pt 1): 135118. DOI: 10.1016/j.ijbiomac.2024.135118.
Doherty AJ, Ashford SR, Subramanya HS, Wigley DB. 1996. Bacteriophage T7 DNA ligase. Overexpression, purification, crystallization, and characterization. J Biol Chem 271 (19): 11083-11089. DOI: 10.1074/jbc.271.19.11083.
Dunne M, Hupfeld M, Klumpp J, Loessner MJ. 2018. Molecular basis of bacterial host interactions by Gram-positive targeting bacteriophages. Viruses 10 (8): 397. DOI: 10.3390/v10080397.
Fymat AL. 2017. Antibiotics and antibiotic resistance. Biomed J Sci Technol Res 1 (1): 65-80. DOI: 10.26717/bjstr.2017.01.000117.
Göller PC, Elsener T, Lorgé D, Radulovic N, Bernardi V, Naumann A, Amri N, Khatchatourova E, Coutinho FH, Loessner MJ, Gómez-Sanz E. 2021. Multi-species host range of staphylococcal phages isolated from wastewater. Nat Commun 12 (1): 6965. DOI: 10.1038/s41467-021-27037-6.
Huang C. 2020. Diabetic foot ulcer with Alcaligenes faecalis infection. Dubai Diabet Endocrinol J 26 (3): 1-6. DOI: 10.1159/000508094.
Hyman P, Abedon ST. 2010. Bacteriophage host range and bacterial resistance. Adv Appl Microbiol 70: 217-248. DOI: 10.1016/S0065-2164(10)70007-1.
IDF [International Diabetes Federation]. 2024. IDF Diabetes Atlas (12th eds). https://diabetesatlas.org/ [Last accessed: 05/04/2025].
Indonesian Ministry of Health. 2023. Cegah sebelum terlambat: Diabetic foot ulcer. Available from: https://yankes.kemkes.go.id/view_artikel/ 2759/cegah-sebelum-terlambat-diabetic-foot-ulcer. [5 April 2023]. [Indonesian]
Jamal M, Hussain T, Das CR, Andleeb S. 2016. Characterization of Siphoviridae phage Z and studying its efficacy against multidrug-resistant Klebsiella pneumoniae planktonic cells and biofilm. J Med Microbiol 64 (Pt 4): 454-462. DOI: 10.1099/jmm.0.000040.
Kawasaki T, Narulita E, Matsunami M, Ishikawa H, Shimizu M, Fujie M, Bhunchoth A, Phironrit N, Chatchawankanphanich O, Yamada T. 2016. Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum. Virology 492: 73-81. DOI: 10.1016/j.virol.2016.02.011.
Legotsky SA, Vlasov KY, Priyma AD, Shneider MM, Pugachev VG, Totmenina OD, Kabanov AV, Miroshnikov KA, Klyachko NL. 2014. Peptidoglycan-degrading activity of the broad-range Salmonella bacteriophage S-394 recombinant endolysin. Biochimie 107 Pt B: 293-299. DOI: 10.1016/j.biochi.2014.09.017.
Li XX, Chen Y, Wang S, Duan X, Zhang F, Guo A, Tao P, Chen H, Li X, Qian P. 2022. Exploring the benefits of metal ions in phage cocktails for the treatment of Methicillin-Resistant Staphylococcus aureus (MRSA) infection. Infect Drug Resist 15: 2689-2702. DOI: 10.2147/idr.s362743.
Lin DM, Koskella B, Lin HC. 2017. Phage therapy: An alternative to antibiotics in the age of multidrug resistance. World J Gastrointest Pharmacol Ther 8 (3): 162-173. DOI: 10.4292/wjgpt.v8.i3.162.
Lingga R, Budiarti S. 2023. Bacteriophage and its potential application in wastewater treatment. Trop Microb J 1 (1): 25-34. DOI: 10.24246/tmj.v1i1.10604. [Indonesian]
Mavrich TN, Hatfull GF. 2017. Bacteriophage evolution differs by host, lifestyle, and genome. Nat Microbiol 2: 17112. DOI: 10.1038/nmicrobiol.2017.112.
Mayrovitz HN, Wong S, Mancuso C. 2023. Venous, arterial, and neuropathic leg ulcers with emphasis on the geriatric population. Cureus 15 (4): e38123. DOI: 10.7759/cureus.38123.
Nale JY, Al-Tayawi TS, Heaphy S, Clokie MRJ. 2021. Impact of phage CDHS-1 on the transcription, physiology, and pathogenicity of Clostridioides difficile ribotype 027 strain R20291. Viruses 13 (11): 2262. DOI: 10.3390/v13112262.
Narulita E, Cahyati VIN, Febrianti RA, Iqbal M. 2023. Potential bacteriophages to overcome bacterial infection of Alcaligenes faecalis in diabetic ulcer. Pediatr Endocrinol Diabetes Metab 29 (2): 61-66. DOI: 10.5114/pedm.2023.125363.
Narulita E, Ramadhan F, Febrianti RAF, Yulian R, Rofiqoh A, Firdausy ZA, Iqbal M. 2025. Partial characterization of bacteriophages infecting Salmonella sp. cause of foodborne disease. J Microbiol Biotechnol Food Sci 14 (5): e10240. DOI: 10.55251/jmbfs.10240.
Naureen Z, Dautaj A, Anpilogov K, Camilleri G, Dhuli K, Tanzi B, Maltese PE, Cristofoli F, De Antoni L, Beccari T, Dundar M, Bertelli M. 2020. Bacteriophages presence in nature and their role in the natural selection of bacterial populations. Acta Biomed 91 (13-S): e2020024. DOI: 10.23750/abm.v91i13-s.10819.
Qin S, Xiao W, Zhou C, Pu Q, Deng X, Lan L, Liang H, Song X, Wu M. 2022. Pseudomonas aeruginosa: Pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances, and emerging therapeutics. Signal Transduct Target Ther 7 (1): 199. DOI: 10.1038/s41392-022-01056-1.
Ranveer SA, Dasriya V, Ahmad MF et al. 2024. Positive and negative aspects of bacteriophages and their immense role in the food chain. NPJ Sci Food 8 (1): 1. DOI: 10.1038/s41538-023-00245-8.
Risqiyah W, Narulita E, Rofiqoh A, Ludfi AS, Iqbal M. 2022. Morphological and molecular identification of multi-antibiotic-resistant bacteria in the wound site of diabetic ulcers. Biodiversitas 23 (2): 663-670. DOI: 10.13057/biodiv/d230207.
Ross A, Ward S, Hyman P. 2016. More is better: Selecting for broad host range bacteriophages. Front Microbiol 7: 1352. DOI: 10.3389/fmicb.2016.01352.
Sadigh-Eteghad S, Dehnad A, Mahmodi J, Hoseyni H, Khalili I, Razmaray 2nd N. 2013. Healing potential of a Streptomyces sp. secondary metabolite, SEM-1-111, on experimental full-thickness excision cutaneous wounds in Wistar rats. Clin Exp Dermatol 38 (2): 178-184. DOI: 10.1111/ced.12026.
Samia AS. 2024. Effect of Penicillium species on the antibiotic resistance profile of Alcaligenes faecalis. Afr J Infect Dis 18 (2): 8-18. DOI: 10.21010/ajidv18i2.2.
Taha OA, Connerton PL, Connerton IF, El-Shibiny A. 2018. Bacteriophage ZCKP1: A potential treatment for Klebsiella pneumoniae isolated from diabetic foot patients. Front Microbiol 9: 2127. DOI: 10.3389/fmicb.2018.02127.
Tan CW, Rukayadi Y, Hasan H, Abdul-Mutalib N-A, Jambari NN, Hara H, Thung TY, Lee E, Radu S. 2021. Isolation and characterization of six Vibrio parahaemolyticus lytic bacteriophages from seafood samples. Front Microbiol 12: 616548. DOI: 10.3389/fmicb.2021.616548.
Ul Haq I, Chaudhry WN, Akhtar MN, Andleeb S, Qadri I. 2012. Bacteriophages and their implications on future biotechnology: A review. Virol J 9: 9. DOI: 10.1186/1743-422x-9-9.
Wangchuk J, Chatterjee A, Patil S, Madugula SK, Kondabagil K. 2021. The coevolution of large and small terminases of bacteriophages is a result of purifying selection leading to phenotypic stabilization. Virology 564: 13-25. DOI: 10.1016/j.virol.2021.09.004.
Yang S, Hu L, Zhao Y, Meng G, Xu S, Han R. 2024. Prevalence of multidrug-resistant bacterial infections in diabetic foot ulcers: A meta-analysis. Intl Wound J 21 (4): e14864. DOI: 10.1111/iwj.14864.
Zhang C, Quan X, Lian W, Liu R, Wen Q, Chen X. 2024. Phenotypic characterization and genomic analysis of Limosilactobacillus fermentum phage. Curr Res Food Sci 8: 100748. DOI: 10.1016/j.crfs.2024.100748.