Increasing bacterial resistance has led to an alarming rate of nosocomial infection leading to a call for alternative antibiotics [1]. Specifically, catheter-associated urinary tract infections (CAUTIs) are costly, affect 450,000 patients per year [2] and do not have suitable antimicrobial coatings to combat resistance. Treatments such as silver are cytotoxic and only short-term. AMPs such as Chrysophsin-1 (CHY1) are promising alternatives, as they are naturally-derived, broad-spectrum and have unique mechanisms making resistance much less likely [3]-[6]. AMP surface tethering may provide stable delivery, but immobilization may reduce activity [5]. Previously, we used quartz crystal microbalance with dissipation (QCM-D) to propose mechanisms of cysteine-modified CHY1 (C-CHY1) when covalently bound via a short and long PEG tether lengths (molecular weight 866 and 7500) onto SiO2. C-CHY1 activity was independent of bacteria type (gram negative vs. positive), but tether length caused changes in mechanisms: PEG866 forced a non-native (yet still potent) ion-displacement mechanism while the highest activity was achieved with PEG7500 natural pore formation. Thus, our goal was to immobilize C-CHY1 onto a catheter material via PEG 866 and 7500 to confirm QCM-D findings ex situ and allow for more rational design of catheter materials to prevent CAUTIs.
Methods: Synthetic CHY1 was covalently tethered to sterile 1cm sections of a BARD silicon catheter by submerging in (1) 10% 3-(aminopropyl)trimethoxysilane (APTMS) for 20min, (2) methanol and water, (3) 100uM PEG 866 or 7500 in PBS for 30min (4) PBS, (5) 10uM C-CHY1 in PBS for 30min, (6) PBS and (7) 106 cfu/mL P. aeruginosa (PA) or gentamycin- and methicillin-resistant S. aureus (MRSA). Absorbance (590nm) was measured over time. For comparison, APTMS-only (steps 1-2, 6-7) and untreated surfaces (steps 6-7) were tested. Activity was determined (n=4 replicates) by plotting A590 vs. time to show relative growth rates.
Results and Discussion: Previously, C-CHY1 tethered via PEG866 and PEG7500 achieved an average killing of 48.7±3.4% and 59.7±3.1%, respectively, which were 1.6 and 2.0 times higher than the control. The growth rates during logarithmic growth of PA and MRSA for PEG866-tethered C-CHY1 were 0.0276 and 0.0448, respectively, and were 0.0273 and 0.103, respectively for PEG7500-tethered C-CHY1. The bacterial growth rate for PEG 866 treated catheter was reduced significantly for both PA and MRSA ~2.5 times slower than the bare surface. Compared to the bare surface growth of PA was 3.6 times slower for PEG7500 but 55% lower against MRSA. This indicates that PEG866 is more broad-spectrum on catheter surfaces than SiO2. PEG7500 is much more effective against gram negative microbes on catheters than is PEG866, but is only broad-spectrum on SiO2. Together, these results suggest that the activity of tethered C-CHY1 is dependent not only on tether length, which may change the mechanism, but also on the type of immobilization surface.
References:
[1] CDC Antibiotic resistant threats in the United States, 2013
[2] CDC The direct medical costs of healthcare-associated infections in US hospitals and the benefits of prevention, 2009
[3] Kazemzadeh-Narbat M. Biomaterials 2010:31:9519:9526
[4] Hickok NJ. Adv Drug Deliv Rev. 2012:64:1165-1176
[5] Bagheri M. Anitmicrob Agents Chemother. 2009:53:1132-1141
[6] Ivanov IE. ACS Appl Mater Interfaces 2012:4:5891-5897