en Anti-microbial agents, resistance and treatment protocols Anti-microbial agents, resistance and treatment protocols Original article Original article <div style="text-align: justify;"><b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">Introduction: </span></span></span></b><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a major public health concern due to its multidrug resistance and robust biofilm formation, which contribute to persistent infections and treatment failures. Novel antibiofilm agents are urgently needed to combat drug-resistant pathogens. This study evaluated the antibacterial and antibiofilm efficacy of NFPPO, a novel nitrofuranyl pyranopyrimidinone derivative, against preformed 1-, 3-, and 5-day-old MRSA biofilms. <b>Methods:</b></span></span></span> <span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times=""><span style="color:black">NFPPO was synthesized via a tandem Knoevenagel-Michael cyclocondensation reaction.<b>&nbsp;</b>The minimum inhibitory concentration (MIC) of NFPPO was determined against four clinical MRSA isolates and the reference strain&nbsp;<i>S. aureus</i>&nbsp;ATCC 700699.<b>&nbsp;</b>Antibiofilm activity was assessed against preformed 1-, 3-, and 5-day-old MRSA biofilms using the crystal violet assay.<b>&nbsp;</b>Confocal laser scanning microscopy (CLSM) quantified structural and viability changes in preformed biofilms post-NFPPO treatment.<b> Results: </b></span></span></span></span><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">All tested MRSA isolates and <i>S. aureus</i> ATCC 700699 exhibited vancomycin resistance (classified as VRSA), whereas NFPPO showed potent antibacterial activity (MIC, 8&ndash;16 &micro;g/mL). All isolates formed robust biofilms (OD625 &ge; 3.0). Crystal violet assays and CLSM demonstrated significant NFPPO antibiofilm activity, reducing biofilm biomass and viability</span></span></span><span style="font-size:10.0pt"><span style="line-height:107%"><span new="" roman="" style="font-family:" times="">. <b>Conclusion:</b> NFPPO exhibited superior antibiofilm efficacy compared to vancomycin in eradicating <i>in vitro</i> preformed 1-, 3-, and 5-day-old MRSA biofilms. These findings position NFPPO as a promising candidate for further development to target biofilm-associated MRSA infections<span style="background:white">.</span></span></span></span></div> Methicillin-resistant Staphylococcus aureus, pyranopyrimidinone, antibiofilm agents, confocal laser scanning microscopy 139 146 http://jommid.pasteur.ac.ir/browse.php?a_code=A-10-441-8&slc_lang=en&sid=1 Sepideh Soltani soltanisepideh99@gmail.com 0009-0003-0714-703X No Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran Nasrin Saberi Harooni 0000-0003-0309-6522 No Medicinal Chemistry Laboratory, Department of Clinical Research, Pasteur Institute of Iran, Tehran, Iran Fatemeh Dehghani Tafti 0009-0006-3015-4774 No Medicinal Chemistry Laboratory, Department of Clinical Research, Pasteur Institute of Iran, Tehran, Iran Morvarid Shafiei dr.shafiei80@gmail.com 0000-0002-6166-5028 Yes Department of Bacteriology, Pasteur Institute of Iran, Tehran, Iran Azar Tahghighi atahghighi2009@gmail.com 0000-0002-1221-4490 No Medicinal Chemistry Laboratory, Department of Clinical Research, Pasteur Institute of Iran, Tehran, Iran