Antibiofilm Effect of Poly(Vinyl Alcohol- co-Ethylene) Halamine Film against Listeria innocua and Escherichia coli O157:H7

Abstract

Bacterial biofilm formation is linked to several infections and foodborne disease outbreaks. To address this challenge, there is an unmet need to develop rechargeable antimicrobial materials that can provide continuous sanitation of contact surfaces, especially in the food industry. This study was aimed at evaluating a novel rechargeable antimicrobial polymer formed using poly(vinyl alcohol-co-ethylene) (PVA-co-PE) with halamine functionality to prevent biofilm formation with repeated exposure to high loads of bacteria and organic content and also to aid in inactivation of preformed biofilms upon contact with this novel material. The antibiofilm activity of this rechargeable antimicrobial material was evaluated using a combination of fluorescence and scanning electron microscopy techniques and biofilm metabolic activity analyses. The results determined on the basis of imaging and metabolic activity measurements demonstrated that halamine-functionalized polymer films significantly reduced Listeria innocua and Escherichia coli O157:H7 biofilm formation. This novel polymeric material maintained its antibiofilm activity with repeated cycles of extended exposure to high levels of bacterial load. These polymeric films were recharged using bleach and cleaned using mechanical sonication after each cycle of extended incubation with bacteria. Halamine-functionalized polymeric material also exhibited significant antibacterial activity against preformed biofilms on a model surface. In summary, our results demonstrate the potential of this antimicrobial material to provide continuous sanitation of surfaces and applications for inactivating preformed biofilms without extensive use of resources, including water and heat. This polymeric material may be used as a replacement for existing polymeric materials or as a coating on diverse materials.IMPORTANCE Conventional sanitizers can have limited efficacy in inactivating biofilms in areas with limited accessibility and buildup of organic biomass. Furthermore, none of the current approaches provide continuous sanitation of surfaces. There is a significant unmet need to develop and validate materials that can prevent biofilm formation as well as inactivate preformed biofilms. In this study, the efficacy of a copolymer film containing N-halamine against biofilms of L. innocua and E. coli O157:H7 was evaluated. The polymer film showed strong inhibitory activity against pregrown biofilm or prevented the growth of a new biofilm. The polymer film also maintained its antibiofilm activity after multiple cycles of exposure to high titers of bacterial load with recharging of the polymer film using bleach at intermediate steps between the cycles. Overall, the results demonstrate the potential of a novel antimicrobial material to inhibit and treat biofilms in food industry applications.

Keywords: Escherichia coli O157:H7; Listeria innocua; antimicrobial polymers; biofilm; halamines; sanitation.

FIG 1

Fluorescence microscopy of bacterial biofilm growth on PVA-co-PE films. (A and B) Images show calcofluor staining of L. innocua (A) and E. coli O157:H7 (B) biofilms after 24 h of biofilm growth on the surface of control films (CTRL FILM; not charged) or N-halamine-charged films (HALA FILM) of 0.1-, 0.5-, or 1-mm thickness. Bright field (BF) and fluorescence (FL) images are shown for each sample. (C) Images of the 1-mm-thick films without biofilms and without calcofluor.

FIG 2

Resazurin metabolic activity assay of bacterial biofilm on PVA-co-PE films. Graphs show the fluorescent intensity curves of resazurin reduction as a function of the time (in minutes) for L. innocua biofilms grown on CTRL (blue) or HALA (red) films of 0.1 (A)-, 0.5 (B)-, and 1 (C)-mm thickness and E. coli O157:H7 biofilms grown on CTRL or HALA films of 0.1 (D)-, 0.5 (E)-, and 1 (F)-mm thickness. Curves for HALA films without biofilm (green) are also shown. The numbers on the curves indicate the times (in minutes) required to reach the peak fluorescence signal intensity corresponding to resazurin reduction.

FIG 3

Antibiofilm properties of recharged HALA films with multiple cycles of biofilm growth assessed using the FDA enzymatic activity assay. Graphs show the relative fluorescent intensities (expressed as percentages) of the FDA deesterification product after 30 min of incubation to assess the enzymatic activity of bacterial cells in a biofilm. These measurements were conducted for three cycles of L. innocua (left) and E. coli O157:H7 (right) biofilm growth on CTRL (blue) or HALA (red) films of 1-mm thickness. The films were recharged with bleach after each cycle and reinoculated to grow biofilms. Percentages were calculated based on the fluorescence intensity corresponding to the enzymatic activity of the biofilm on the CTRL film for each cycle. Curves for HALA films without biofilm (green) are also shown, to report the background noise of the assay. Statistical significance was determined (*, P < 0.05; **, P < 0.01).

FIG 4

Fluorescence microscopy of bacterial biofilm growth on recharged HALA films with multiple cycles of biofilm growth. The calcofluor staining of L. innocua (A) and E. coli O157:H7 (B) biofilms after 24 h of biofilm growth on the surface of CTRL or recharged HALA film of 1-mm thickness is shown. Bright field (BF) and fluorescence (FL) images are shown for each sample.

FIG 5

SEM images of bacterial biofilm growth on recharged HALA films with multiple cycles of biofilm growth. The figure shows images of L. innocua (A) and E. coli O157:H7 (B) biofilms on the surface of CTRL or recharged HALA film of 1-mm thickness.

FIG 6

Inhibition of the enzymatic activity of biofilm on a plastic surface by contact with HALA films. Graphs show the relative fluorescent intensities (expressed as percentages) of the FDA deesterification product after 30 min of incubation of the L. innocua (left) and E. coli O157:H7 (right) biofilms on a plastic surface upon contact with CTRL or HALA films of 3 different thickness levels (0.1, 0.5, and 1 mm) for 1 h. Percentages were calculated on the basis of the fluorescence intensity corresponding to the enzymatic activity of the biofilm on a plastic surface not treated with any film contact. Statistically significant differences relative to the positive-control sample (BIO+ FDA+) were determined (**, P < 0.01).

FIG 7

SEM images of bacterial biofilm on a plastic surface after contact with HALA film. The figure shows images of L. innocua (A) and E. coli O157:H7 (B) biofilms on a plastic surface after contact with CTRL or HALA films of 0.1- and 1-mm thickness for 1 h. Insets show zoomed sections of the images.