Photoinactivation of Multidrug-Resistant mcr-1-Positive E. coli Using PCPDTBT Conjugated Polymer Nanoparticles under White Light

Abstract

The issue of antimicrobial resistance is an escalating concern within the scope of global health. It is predicted that the existence of antibiotic-resistant bacteria might result in an estimated annual death of up to 10 million by 2050, along with possible economic losses ranging from 100 to 210 trillion. This study reports the production of poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b']dithiophene)-alt-4,7(2,1,3-benzothiadiazole)] nanoparticles (PCPDTBT-NPs) by nanoprecipitation as an alternative to tackle this problem. The size, shape, and optical features of these conjugated polymer NPs were analyzed. Their efficacy as photosensitizers against nonresistant (ATCC) and multidrug-resistant mcr-1-positive Escherichia coli was assessed under white light doses of 250 and 375 J·cm^-2. PCPDTBT-NPs inactivated both E. coli strains exposed to white light at an intensity of 375 J·cm^-2, while no antimicrobial effect was observed in the group not exposed to white light. Reactive oxygen species and singlet oxygen were detected using DCFH-DA and DPBF probes, allowing the investigation of the photoinactivation pathways. This work showcases PCPDTBT-NPs as photosensitizers to eliminate multidrug-resistant bacteria through photodynamic inactivation employing visible light.

Keywords: PCPDTBT; antimicrobial resistance; conjugated polymer; mcr-1 positive Escherichia coli; nanoparticle; photodynamic inactivation.

Figure 1

Schematic diagram of the PCPDTBT nanoparticles production by nanoprecipitation method.

Figure 2

(a) UV–vis absorption spectrum of PCPDTBT-NPs and visual appearance of the solution; (b) Fluorescence spectrum of PCPDTBT-NPs under 405 nm excitation and image displaying the fluorescence in the aqueous solution with PCPDTBT at 17 μg·mL^–1.

Figure 3

(a) Hydrodynamic diameter distribution, ζ potential, and polydispersity index (PDI) from dynamic light scattering measurements and (b) Particle diameter distribution and scanning electron micrography of PCPDTBT-NPs.

Figure 4

(a) UV–vis absorption spectrum of PCPDTBT-NPs as a function of the white light illumination and (b) visual appearance of the solution before and after 90 min of illumination.

Figure 5

UV–vis absorption of PCPDTBT-NPs at 405 nm as a function of the storage time. Inset: visual appearance of the solution after 20 days.

Figure 6

(a) Absorbance of DPBF (0.077 mM) in solution with and without PCPDTBT-NPs (8.5 μg·mL^–1) as a function of time (625 nm irradiation, 3.5 mW), (b) A/A₀ over time, where A₀ and A is the time-zero absorption and the absorption at 415 nm, respectively, (c) Time-dependent fluorescence spectra of DCFH-DA (0.35 mM) in solution with and without PCPDTBT-NPs (8.5 μg·mL^–1) under 625 nm irradiation (3.5 mW), and (d) F/F₀ ratio over time, where F₀ and F is the time-zero fluorescence and the fluorescence at 520 nm, respectively.

Figure 7

Growth of E. coli (ATCC 25922) and mcr-1 positive E. coli (CCBH 23595) colonies with (a) 0.0, (b) 4.25, (c) 8.5, and (d) 17 μg·mL^–1 of PCPDTBT-NPs on plate count agar. The bacterial media were nonirradiated and white light irradiated with energy doses of 250 and 375 J·cm^–2 for 60 and 90 min, respectively.

Figure 8

Mean CFU·mL^–1 of E. coli (ATCC) and mcr-1 positive E. coli (CCBH 23593) subjected to PCPDTBT-NPs. The irradiated group underwent white light illumination with energy doses of 250 and 375 J·cm^–2. The asterisks (*) indicates a significant difference at a 95% confidence level (p < 0.05), whereas the hash symbol (#) denotes that no bacterial colony was observed.

Figure 9

Representative SEM image of E. coli exposed to PCPDTBT-NPs at a concentration of 17 μg·mL^–1: (a) nonilluminated and (b) after white light illumination (250 J·cm^–2). Red arrows show partial or complete damage to the cell wall. Confocal fluorescence image of E. coli subjected to PCPDTBT-NPs at 17 μg·mL^–1: (c) nonilluminated and (d) after white light illumination (250 J·cm^–2). The blue dots represent the PCPDTBT-NPs fluorescence.