Abstract

Biofilms are complex and highly resistant microbial communities that have been found in different chronic infections (1,2). One of the main mechanisms of antibiotic resistance of biofilms is the formation of an extracellular matrix that protects the community. Tobramycin (TOB) is one of the first line antibiotic against Pseudomonas aeruginosa (PA) biofilm infections in cystic fibrosis patients (3,4). However, it has been reported that the activity of TOB against PA biofilms is reduced due to an interaction with the anionic components of the biofilm matrix (5). A previous study showed that the conjugation of TOB with polyethylene glycol (PEG-TOB) resulted in an improved activity against PA biofilms compared to unmodified TOB (6). The purpose of this study was to optimize the synthesis of PEG-TOB by testing different methods and then evaluate the antimicrobial effect of the against PA biofilms. First, we functionalize PEG with succinic acid according to Atassi and Manshouri (1991) with few modifications. Then this acid-PEG (mPEG-COOH) was conjugated to TOB by three different methods: Method A was described by Du, Et al. (2015), and methods B and C were adaptations from Hermanson (2013) and Ferguson, Et al. (2014), respectively. For the three methods, we used tobramycin sulfate, acid-PEG (5,100 kDa) and EDC·HCL and NHS as coupling agents. mPEG-COOH and PEG-TOB were analyzed by proton nuclear magnetic resonance (1H NMR) (Agilent MR 400, 400 MHz, Agilent Technologies, Inc. CA, USA). For in vitro evaluation, Pseudomonas aeruginosa strain PAO1 (ATCC® 15692TM) was grown in BHI medium from an overnight LB agar subculture. The bacterial suspension was adjusted to a concentration of 3 x 107 CFU/mL. For biofilm formation, 100 µL of this suspension were added to each well of 96-well plate and incubated at 37°C and 75 rpm. After 1.5 h of incubation, the wells were rinsed with PBS before the addition of 200 L of fresh media and incubation for 24 h. Different concentrations of TOB and PEG-TOB’s (2.1 – 8.6 – 34.2 – 136.9 M) were used to treat the pre-formed biofilms for 24 h. An XTT assay was used to evaluate the susceptibility of the PAO1 biofilms to the treatment. PEG-TOBB caused the highest reduction in proliferation at 136.9 M (2.66 ± 0.92%) as shown by the XTT assay. It was followed by PEGTOBA (6.52 ± 3.51%) and unmodified TOB (12.03 ± 6.53%). PEG-TOB obtained by method C was not better than TOB alone (40.95 ± 19.9%). The MIC80 of PEG-TOBB was reached with 34.2 M, four times lower than the MIC80 of TOB (34.2 M versus 136.9 M). The results of the XTT assay confirmed a lower degree of conjugation achieved by method C. The 1H NMR analysis showed that the integration values of TOB peak in the PEG-TOB products had a good correlation with the antibiofilm activity of the conjugate. In our study, we confirmed that the pegylation of TOB by 2 different methods resulted in improved antimicrobial activity against PAO1 biofilms. PEG-TOB was able to reduce bacterial proliferation in an 80% with 4-times less concentration compared to unmodified TOB. We found that method B can produce a PEG-TOB with a slightly better activity than our previous report but only within 2 hours of reaction instead of 48 h with method A. 1H NMR can be used to characterize and estimate the degree of conjugation of the PEG-TOB while a quantification method is not available.