Antibiofilm activity and mode of action of DMSO alone and its combination with afatinib against Gram-negative pathogens Mohd Fakharul Zaman Raja Yahya 1,2 & Zazali Alias 1 & Saiful Anuar Karsani 1 Received: 12 October 2016 /Accepted: 10 May 2017 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2017 Abstract Biofilms are complex microbial communities that tend to attach to either biotic or abiotic surface. Enclosed in a self-produced extracellular polymeric substance (EPS) matrix, the biofilms often cause persistent infections. The objective of this study was to investigate the antibiofilm activity of dimeth- yl sulfoxide (DMSO) and afatinib against Gram-negative pathogens. Test microorganisms used in this study were Escherichia coli ATCC 1299, Pseudomonas aeruginosa ATCC 10145, and Salmonella typhimurium ATCC 14028. Biofilms were developed in 96-well microplate at 37°C for 24 h. Following removal of non-adherent cells, analysis of biofilm viability, biofilm biomass, and extracellular polymeric substances (EPS) matrix were performed using resazurin as- say, crystal violet assay, and attenuated total reflectance fouri- er transform infrared (ATR-FTIR) spectroscopy, respectively. Bradford protein assay was conducted to determine the total amount of EPS proteins. The results demonstrated that both 32% DMSO alone and its combination with 3.2 μg/mL afatinib were effective in killing biofilm cells and reducing biofilm biomass. IR spectral variations of EPS matrix of biofilms in the range between 1700 and 900 cm -1 were also observed. Reduction in EPS proteins verified the chemical modifications of EPS matrix. In conclusion, 32% DMSO alone and its combination with 3.2 μg/mL afatinib showed remarkable antibiofilm activities against Gram-negative path- ogens. It was suggested that the biofilm inhibition was medi- ated by the chemical modification of EPS matrix. Keywords Antibiofilm . Dimethyl sulfoxide . Afatinib . Gram-negative pathogen Introduction Biofilms are complex microbial communities that tend to at- tach to either biotic or abiotic surface. Enclosed in a self- produced extracellular polymeric substance (EPS) matrix, the biofilms differ from their planktonic counterparts in the protein expression patterns resulting in distinct metabolism (Giaouris et al. 2013). The bacterial cells within biofilm be- come physiologically heterogeneous due to spatial location and chemical gradient of oxygen, nutrients, waste products, and signaling molecules (Stewart and Franklin 2008). The nature of these phenomena typically results from the forma- tion of a biphasic biofilm consisting a flat-cell monolayer biofilm intermixed with three-dimensional mushroom-shaped biofilm which is different in biomass, surface coverage, and average thickness (Entcheva-Dimitrov and Sporman 2004; Karatan and Watnick 2009). Across many studies, most of biofilm control strategies have focused on prevention of bio- film formation rather than biofilm removal. A broad spectrum of existing therapeutic agents has been extensively studied for prevention of biofilm formation. These include small molecules (Nguyen et al. 2012), acetic acid (Halstead et al. 2015), and silver particle (Sharma et al. 2015). Among the widely used therapeutic agents whose antibiofilm properties have not yet been elucidated are di- methyl sulfoxide (DMSO) and afatinib. DMSO is a naturally * Mohd Fakharul Zaman Raja Yahya fakharulzaman@salam.uitm.edu.my * Saiful Anuar Karsani saiful72@um.edu.my 1 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 2 Faculty of Applied Sciences, Universiti Teknologi MARA Shah Alam, 40450 Shah Alam, Malaysia Folia Microbiol DOI 10.1007/s12223-017-0532-9