In this study, we have focused on a multiparametric microbiological analysis of the antistaphylococcal action of the iodinated imine BH77, designed as an analogue of rafoxanide. Its antibacterial activity against five reference strains and eight clinical isolates of Gram-positive cocci of the genera Staphylococcus and Enterococcus was evaluated. The most clinically significant multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant S. aureus (VRSA), and vancomycin-resistant Enterococcus faecium, were also included. The bactericidal and bacteriostatic actions, the dynamics leading to a loss of bacterial viability, antibiofilm activity, BH77 activity in combination with selected conventional antibiotics, the mechanism of action, in vitro cytotoxicity, and in vivo toxicity in an alternative animal model, Galleria mellonella, were analyzed. The antistaphylococcal activity (MIC) ranged from 15.625 to 62.5 μM, and the antienterococcal activity ranged from 62.5 to 125 μM. Its bactericidal action; promising antibiofilm activity; interference with nucleic acid, protein, and peptidoglycan synthesis pathways; and nontoxicity/low toxicity in vitro and in vivo in the Galleria mellonella model were found to be activity attributes of this newly synthesized compound. In conclusion, BH77 could be rightfully minimally considered at least as the structural pattern for future adjuvants for selected antibiotic drugs. IMPORTANCE Antibiotic resistance is among the largest threats to global health, with a potentially serious socioeconomic impact. One of the strategies to deal with the predicted catastrophic future scenarios associated with the rapid emergence of resistant infectious agents lies in the discovery and research of new anti-infectives. In our study, we have introduced a rafoxanide analogue, a newly synthesized and described polyhalogenated 3,5-diiodosalicylaldehyde-based imine, that effectively acts against Gram-positive cocci of the genera Staphylococcus and Enterococcus. The inclusion of an extensive and comprehensive analysis for providing a detailed description of candidate compound-microbe interactions allows the valorization of the beneficial attributes linked to anti-infective action conclusively. In addition, this study can help with making rational decisions about the possible involvement of this molecule in advanced studies or may merit the support of studies focused on related or derived chemical structures to discover more effective new anti-infective drug candidates.

Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Hradec Králové Czech Republic

Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Hradec Králové Czech Republic

Charles University Faculty of Pharmacy in Hradec Králové Department of Organic and Bioorganic Chemistry Hradec Králové Czech Republic

ELKH ELTE Research Group of Peptide Chemistry Budapest Hungary

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Barsoumian AE, Mende K, Sanchez CJ, Beckius ML, Wenke JC, Murray CK, Akers KS. 2015. Clinical infectious outcomes associated with biofilm-related bacterial infections: a retrospective chart review. BMC Infect Dis 15:223. doi:10.1186/s12879-015-0972-2. PubMed DOI PMC

Friedman ND, Temkin E, Carmeli Y. 2016. The negative impact of antibiotic resistance. Clin Microbiol Infect 22:416–422. doi:10.1016/j.cmi.2015.12.002. PubMed DOI

Stewart PS, Costerton JW. 2001. Antibiotic resistance of bacteria in biofilms. Lancet 358:135–138. doi:10.1016/s0140-6736(01)05321-1. PubMed DOI

Donlan RM. 2002. Biofilms: microbial life on surfaces. Emerg Infect Dis 8:881–890. doi:10.3201/eid0809.020063. PubMed DOI PMC

Yin W, Wang Y, Liu L, He J. 2019. Biofilms: the microbial “protective clothing” in extreme environments. Int J Mol Sci 20:3423. doi:10.3390/ijms20143423. PubMed DOI PMC

Flemming HC, Wingender J. 2010. The biofilm matrix. Nat Rev Microbiol 8:623–633. doi:10.1038/nrmicro2415. PubMed DOI

Sharma D, Misba L, Khan AU. 2019. Antibiotics versus biofilm: an emerging battleground in microbial communities. Antimicrob Resist Infect Control 8:76. doi:10.1186/s13756-019-0533-3. PubMed DOI PMC

Bjarnsholt T. 2013. The role of bacterial biofilms in chronic infections. APMIS Suppl 2013:1–51. doi:10.1111/apm.12099. PubMed DOI

Lebeaux D, Ghigo J-M, Beloin C. 2014. Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol Mol Biol Rev 78:510–543. doi:10.1128/MMBR.00013-14. PubMed DOI PMC

Chambers HF, DeLeo FR. 2009. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 7:629–641. doi:10.1038/nrmicro2200. PubMed DOI PMC

McGuinness WA, Malachova N, DeLeo FR. 2017. Vancomycin resistance in Staphylococcus aureus. Yale J Biol Med 90:269–281. PubMed PMC

Kaur DC, Chate SS. 2015. Study of antibiotic resistance pattern in methicillin resistant Staphylococcus aureus with special reference to newer antibiotic. J Glob Infect Dis 7:78–84. doi:10.4103/0974-777X.157245. PubMed DOI PMC

World Health Organization. 2017. Prioritization of pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis. World Health Organization, Geneva, Switzerland. https://apps.who.int/iris/handle/10665/311820.

Reddy PN, Srirama K, Dirisala VR. 2017. An update on clinical burden, diagnostic tools, and therapeutic options of Staphylococcus aureus. Infect Dis (Auckl) 10:1179916117703999. doi:10.1177/1179916117703999. PubMed DOI PMC

Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG, Jr. 2015. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev 28:603–661. doi:10.1128/CMR.00134-14. PubMed DOI PMC

Ahmed MO, Baptiste KE. 2018. Vancomycin-resistant enterococci: a review of antimicrobial resistance mechanisms and perspectives of human and animal health. Microb Drug Resist 24:590–606. doi:10.1089/mdr.2017.0147. PubMed DOI

Arias CA, Contreras GA, Murray BE. 2010. Management of multidrug-resistant enterococcal infections. Clin Microbiol Infect 16:555–562. doi:10.1111/j.1469-0691.2010.03214.x. PubMed DOI PMC

Zhang J, Huang H, Zhou X, Xu Y, Chen B, Tang W, Xu K. 2019. N-Benzylanilines as fatty acid synthesis inhibitors against biofilm-related methicillin-resistant Staphylococcus aureus. ACS Med Chem Lett 10:329–333. doi:10.1021/acsmedchemlett.8b00612. PubMed DOI PMC

Gong H-H, Baathulaa K, Lv J-S, Ca G-X, Zhou C-H. 2016. Synthesis and biological evaluation of Schiff base-linked imidazolyl naphthalimides as novel potential anti-MRSA agents. Med Chem Commun (Camb) 7:924–931. doi:10.1039/C5MD00574D. DOI

Krátký M, Dzurková M, Janoušek O, Konečná K, Trejtnar F, Stolaříková J, Vinšová J. 2017. Sulfadiazine salicylaldehyde-based Schiff bases: synthesis, antimicrobial activity and cytotoxicity. Molecules 22:1573. doi:10.3390/molecules22091573. PubMed DOI PMC

Krátký M, Konečná K, Janoušek J, Brablíková M, Janďourek O, Trejtnar F, Stolaříková J, Vinšová J. 2020. 4-Aminobenzoic acid derivatives: converting folate precursor to antimicrobial and cytotoxic agents. Biomolecules 10:9. doi:10.3390/biom10010009. PubMed DOI PMC

Krátký M, Konečná K, Brokešová K, Maixnerová J, Trejtnar F, Vinšová J. 2021. Optimizing the structure of (salicylideneamino)benzoic acids: towards selective antifungal and antistaphylococcal agents. Eur J Pharm Sci 159:105732. doi:10.1016/j.ejps.2021.105732. PubMed DOI

Krátký M, Konečná K, Janoušek J, Janďourek O, Maixnerová J, Kalivodová S, Trejtnar F, Vinšová J. 2021. Sulfonamide-salicylaldehyde imines active against methicillin- and trimethoprim/sulfonamide-resistant staphylococci. Future Med Chem 13:1945–1962. doi:10.4155/fmc-2021-0169. PubMed DOI

Xu S-P, Lv P-C, Shi L, Zhu H-L. 2010. Design, synthesis, and pharmacological investigation of iodined salicylimines, new prototypes of antimicrobial drug candidates. Arch Pharm (Weinheim) 343:282–290. doi:10.1002/ardp.200900129. PubMed DOI

Vasudevan P, Tandon M. 2010. Antimicrobial properties of iodine based products. J Sci Ind Res 69:376–383.

Molchanova N, Nielsen JE, Sørensen KB, Prabhala BK, Hansen PR, Lund R, Barron AE, Jenssen H. 2020. Halogenation as a tool to tune antimicrobial activity of peptoids. Sci Rep 10:14805. doi:10.1038/s41598-020-71771-8. PubMed DOI PMC

Cotsonas King A, Wu L. 2009. Macromolecular synthesis and membrane perturbation assays for mechanisms of action studies of antimicrobial agents. Curr Protoc Pharmacol Chapter 13:Unit 13A.7. doi:10.1002/0471141755.ph13a07s47. PubMed DOI

Cunningham ML, Kwan BP, Nelson KJ, Bensen DC, Shaw KJ. 2013. Distinguishing on-target versus off-target activity in early antibacterial drug discovery using a macromolecular synthesis assay. J Biomol Screen 18:1018–1026. doi:10.1177/1087057113487208. PubMed DOI

Kostakioti M, Hadjifrangiskou M, Hultgren SJ. 2013. Bacterial biofilms: development, dispersal, and therapeutic strategies in the dawn of the postantibiotic era. Cold Spring Harb Perspect Med 3:a010306. doi:10.1101/cshperspect.a010306. PubMed DOI PMC

Ignasiak K, Maxwell A. 2017. Galleria mellonella (greater wax moth) larvae as a model for antibiotic susceptibility testing and acute toxicity trials. BMC Res Notes 10:428. doi:10.1186/s13104-017-2757-8. PubMed DOI PMC

Allegra E, Titball RW, Carter J, Champion OL. 2018. Galleria mellonella larvae allow the discrimination of toxic and non-toxic chemicals. Chemosphere 198:469–472. doi:10.1016/j.chemosphere.2018.01.175. PubMed DOI

OECD. 2022. OECD guidelines for the testing of chemicals. https://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals_72d77764-en. Retrieved 11 September 2022.

World Health Organization. 2015. Global action plan on antimicrobial resistance. World Health Organization, Geneva, Switzerland. https://apps.who.int/iris/handle/10665/193736.

Ventola CL. 2015. The antibiotic resistance crisis. Part 1: causes and threats. P T 40:277–283. PubMed PMC

Rossolini GM, Arena F, Pecile P, Pollini S. 2014. Update on the antibiotic resistance crisis. Curr Opin Pharmacol 18:56–60. doi:10.1016/j.coph.2014.09.006. PubMed DOI

Fair RJ, Tor Y. 2014. Antibiotics and bacterial resistance in the 21st century. Perspect Medicin Chem 6:25–64. doi:10.4137/PMC.S14459. PubMed DOI PMC

Miró-Canturri A, Ayerbe-Algaba R, Villodres AR, Pachón J, Smani Y. 2020. Repositioning rafoxanide to treat Gram-negative bacilli infections. J Antimicrob Chemother 75:1895–1905. doi:10.1093/jac/dkaa103. PubMed DOI

Hill JA, Cowen LE. 2015. Using combination therapy to thwart drug resistance. Future Microbiol 10:1719–1726. doi:10.2217/fmb.15.68. PubMed DOI

Kumar S, Siji JV, Nambisan B, Mohandas C. 2012. Activity and synergistic interactions of stilbenes and antibiotic combinations against bacteria in vitro. World J Microbiol Biotechnol 28:3143–3150. doi:10.1007/s11274-012-1124-0. PubMed DOI

Junqueira JC. 2012. Galleria mellonella as a model host for human pathogens. Virulence 3:474–476. doi:10.4161/viru.22493. PubMed DOI PMC

Matuschek E, Brown DF, Kahlmeter G. 2014. Development of the EUCAST disk diffusion antimicrobial susceptibility testing method and its implementation in routine microbiology laboratories. Clin Microbiol Infect 20:O255–O266. doi:10.1111/1469-0691.12373. PubMed DOI

Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, Beachey EH. 1985. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol 22:996–1006. doi:10.1128/jcm.22.6.996-1006.1985. PubMed DOI PMC

Stepanović S, Vuković D, Hola V, Di Bonaventura G, Djukić S, Cirković I, Ruzicka F. 2007. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS 115:891–899. doi:10.1111/j.1600-0463.2007.apm_630.x. PubMed DOI

National Committee for Clinical Laboratory Standards. 1999. Methods for determining bactericidal activity of antimicrobial agents; approved guideline. M26-A. National Committee for Clinical Laboratory Standards, Wayne, PA.

Haydak MH. 1936. Is wax a necessary constituent of the diet of wax moth larvae? Ann Entomol Soc Am 29:252–253.