Tetrasodium EDTA Is Effective at Eradicating Biofilms Formed by Clinically Relevant Microorganisms from Patients' Central Venous Catheters

Abstract

Central venous access devices (CVADs) are an essential component of modern health care. However, their prolonged use commonly results in microbial colonization, which carries the potential risk of hospital-acquired bloodstream infections. These infections complicate the treatment of already sick individuals and cost the existing health care systems around the world millions of dollars. The microbes that colonize CVADs typically form multicellular biofilms that are difficult to dislodge and are resistant to antimicrobial treatments. Clinicians are searching for better ways to extend the working life span of implanted CVADs, by preventing colonization and reducing the risk of bloodstream infections. In this study, we analyzed 210 bacterial and fungal isolates from colonized CVADs or human bloodstream infections from two hospitals geographically separated in the east and west of Canada and screened the isolates for biofilm formation in vitro Twenty isolates, representing 12 common, biofilm-forming species, were exposed to 4% tetrasodium EDTA, an antimicrobial lock solution that was recently approved in Canada for use as a medical device. The EDTA solution was effective at eradicating surface-attached biofilms from each microbial species, indicating that it could likely be used to prevent biofilm growth within CVADs and to eliminate established biofilms. This new lock solution fits with antibiotic stewardship programs worldwide by sparing the use of important antibiotic agents, targeting prevention rather than the expensive treatment of hospital-acquired infections.IMPORTANCE The colonization of catheters by microorganisms often precludes their long-term use, which can be a problem for human patients that have few body sites available for new catheters. The colonizing organisms often form biofilms, and increasingly these organisms are resistant to multiple antibiotics, making them difficult to treat. In this article, we have taken microorganisms that are associated with biofilm formation in catheters from two Canadian hospitals and tested them with tetrasodium EDTA, a new antimicrobial catheter lock solution. Tetrasodium EDTA was effective at eliminating Gram-positive, Gram-negative, and fungal species and represents a promising alternative to antibiotic treatment with less chance of the organisms developing resistance. We expect that our results will be of interest to researchers and clinicians and will lead to improved patient care.

Keywords: EDTA; Gram-negative; Gram-positive; antibiotic resistance; antimicrobial; biofilms; catheters; central venous access devices; fungi; minimum inhibitory concentration.

Copyright © 2018 Liu et al.

Figures

FIG 1

Biofilm screening of S. epidermidis isolates. Twenty-five S. epidermidis isolates originating from central venous access devices were inoculated into 96-well plates and grown for 24 h at 37°C in biofilm media: M9, M9 minimal media; CAA, Casamino Acids; TSB, tryptic soy broth. Biofilm cell mass in each well was quantitated by crystal violent staining and measuring the absorbance of the resulting solution at 590 nm (A590). Bars represent the average values and error bars the standard deviations from 6 biological replicates. The dashed horizontal line represents the average A590 value from uninoculated control wells. Stars denote isolates that were judged to have robust biofilm formation. Isolate 170 (arrow) was chosen for subsequent testing.

FIG 2

Determination of MIC, MBC, and MBEC values of tetrasodium EDTA against Staphylococcus isolates cultured from central venous access devices. Individual isolates of S. epidermidis (A, B, and C) and methicillin-resistant S. aureus (D, E, and F) were tested in MIC (A and D), MBC (B and E), and MBEC (C and F) assays. Horizontal bars represent the mean OD600 or viable bacterial cell (CFU/ml) values after cultures were exposed to increasing amounts of tetrasodium EDTA. Arrows represent MIC, MBC, and MBEC values. The dashed horizontal lines on each graph represent the background OD600 values in uninoculated control wells (A and D) or the CFU limit of detection (B, C, E, and F). Three biological replicate cultures were tested in duplicate or triplicate for each type of assay; each dot represents one replicate.

FIG 3

Minimum exposure time to kill bacterial/fungal biofilms with 4% tetrasodium EDTA. In vitro biofilms formed by Gram-positive bacteria (A), Gram-negative bacteria (B), fungal species (C), and control bacteria (D) were formed on polystyrene pegs prior to testing. For each graph, the hatched bar (0 h) shows the starting CFU/ml values measured from control pegs (n = 8). Formed biofilms were exposed to 4% tetrasodium EDTA (black bars) or water (gray bars) for the times shown; four biological replicates with four technical replicates (n = 16) were analyzed for treatment groups, along with four biological replicates with two technical replicates (n = 8) for water controls. Bars represent the average CFU/ml detected, and error bars represent the standard deviation. The time points where biofilms were killed near or at the limit of detection (dotted line [125 CFU/ml]) are highlighted in yellow. Values from each treatment group were compared to the corresponding water controls by unpaired t tests with Welch’s correction. Statistical significance is noted above each treatment bar: ns, not significant (P > 0.05); *, P < 0.05. Arrows denote the minimum exposure times required for complete eradication of the bacterial/fungal biofilms.

FIG 3

Minimum exposure time to kill bacterial/fungal biofilms with 4% tetrasodium EDTA. In vitro biofilms formed by Gram-positive bacteria (A), Gram-negative bacteria (B), fungal species (C), and control bacteria (D) were formed on polystyrene pegs prior to testing. For each graph, the hatched bar (0 h) shows the starting CFU/ml values measured from control pegs (n = 8). Formed biofilms were exposed to 4% tetrasodium EDTA (black bars) or water (gray bars) for the times shown; four biological replicates with four technical replicates (n = 16) were analyzed for treatment groups, along with four biological replicates with two technical replicates (n = 8) for water controls. Bars represent the average CFU/ml detected, and error bars represent the standard deviation. The time points where biofilms were killed near or at the limit of detection (dotted line [125 CFU/ml]) are highlighted in yellow. Values from each treatment group were compared to the corresponding water controls by unpaired t tests with Welch’s correction. Statistical significance is noted above each treatment bar: ns, not significant (P > 0.05); *, P < 0.05. Arrows denote the minimum exposure times required for complete eradication of the bacterial/fungal biofilms.

FIG 3

Minimum exposure time to kill bacterial/fungal biofilms with 4% tetrasodium EDTA. In vitro biofilms formed by Gram-positive bacteria (A), Gram-negative bacteria (B), fungal species (C), and control bacteria (D) were formed on polystyrene pegs prior to testing. For each graph, the hatched bar (0 h) shows the starting CFU/ml values measured from control pegs (n = 8). Formed biofilms were exposed to 4% tetrasodium EDTA (black bars) or water (gray bars) for the times shown; four biological replicates with four technical replicates (n = 16) were analyzed for treatment groups, along with four biological replicates with two technical replicates (n = 8) for water controls. Bars represent the average CFU/ml detected, and error bars represent the standard deviation. The time points where biofilms were killed near or at the limit of detection (dotted line [125 CFU/ml]) are highlighted in yellow. Values from each treatment group were compared to the corresponding water controls by unpaired t tests with Welch’s correction. Statistical significance is noted above each treatment bar: ns, not significant (P > 0.05); *, P < 0.05. Arrows denote the minimum exposure times required for complete eradication of the bacterial/fungal biofilms.