Role of antibiofilm-antimicrobial agents in controlling device-related infections

Abstract

Objectives: To assess the effects of N-acetylcysteine (NAC) on organism viability in planktonic and biofilm phases, biofilm thickness, and extracellular polysaccharide content.

Methods: We performed time-kill curves and broth macrodilution assays of bacterial and fungal clinical isolates with varying concentrations of NAC. We also created in vitro bacterial biofilms, incubated them with NAC or control, and then stained with propidium iodide and FITC-labeled concanavalin A. We measured biofilm thickness, number of non-viable cells, and fluorescent intensity as a marker of extracellular matrix via a confocal laser scanning microscope. All experiments were conducted in triplicate. Tested organisms included methicillin-sensitive and -resistant Staphylococcus aureus (MSSA, MRSA), S. epidermidis, vancomycin-resistant Enterococcus faecalis (VRE), Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae, Candida albicans and C. krusei.

Results: NAC 80 mg/ml was uniformly bactericidal (>99.9% reduction) against all tested bacteria with no recoverable organisms after 30 minutes of incubation, but was fungistatic against candida species. Minimum inhibitory and bactericidal concentrations of NAC ranged from 5-10 mg/ml. Biofilm thickness was significantly decreased in NAC-treated biofilms for all organisms except VRE. The number of non-viable cells in NAC-treated Gram-positive biofilms was increased (p<0.05 for MRSA and VRE). NAC-treated Gram-negative biofilms had scant cellularity and lacked complex 3-dimensional structures that were characteristic of controls. Fluorescent intensity was similar in the experimental and control arms.

Conclusions: NAC is bactericidal against clinically relevant and drug-resistant bacteria and also leads to biofilm disruption. NAC has the potential for use as a novel agent for prevention or treatment of biofilm-related infections.

Figure 1

Time-kill curves of tested organisms with N-acetylcysteine (NAC). The y-axis denotes the mean CFU/ml and error bars signify standard deviation.

Figure 2

Z-stack images of Pseudomonas aeruginosa in control group (top panel) and NAC-treated group (lower panel).

Figure 3

Mean biofilm thickness in µm of 24-hour biofilms of bacterial species when exposed to tryptic soy broth (TSB) alone as control or TSB containing 80 mg/ml of N-acetylcysteine (NAC). Thickness was measured by means of Z-stack imaging with a confocal laser scanning microscope. All differences between individual control and NAC-treated biofilms were statistically significant (p<0.05), except for VRE. MRSA – methicillin-resistant Staphyloccus aureus MSSA – methicillin-sensitive S. aureus VRE – vancomycin-resistant Enterococcus faecalis

Figure 4

Confocal images of P. aeruginosa control (top left), NAC-treated (top right) and MRSA control (bottom left) and NAC-treated (bottom right) utilizing a 60× dipping lens. The scale bar represents 10 µm.

Figure 5

Mean number of non-viable bacterial cells (stained red with propidium iodide when viewed via microscopy) in the control biofilm and NAC-treated biofilm. *Denotes p<0.05. MRSA – methicillin-resistant Staphyloccus aureus MSSA – methicillin-sensitive S. aureus VRE – vancomycin-resistant Enterococcus faecalis