Low concentration of ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria monocytogenes by inhibiting its initial adherence

Food Microbiology 29 (2012) 10e17

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Low concentration of ethylenediaminetetraacetic acid (EDTA) affects biofilm formation of Listeria monocytogenes by inhibiting its initial adherence Yuhua Chang a, b, Weimin Gu c, Lynne McLandsborough a, * a

Department of Food Science, University of Massachusetts, 102 Holdsworth Way, Amherst, MA 01003, USA College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, Shaanxi 710062, PR China c Department of Microbiology, University of Massachusetts, Amherst, MA 01003, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2011 Received in revised form 15 July 2011 Accepted 16 July 2011 Available online 23 July 2011

The distribution and survival of the food-borne pathogen Listeria monocytogenes is associated with its biofilm formation ability, which is affected by various environmental factors. Here we present the first evidence that EDTA at low concentration levels inhibits the biofilm formation of L. monocytogenes. This effect of EDTA is not caused by a general growth inhibition, as 0.1 mM EDTA efficiently reduced the biofilm formation of L. monocytogenes without affecting the planktonic growth. Adding 0.1 mM of EDTA at the starting time of biofilm formation had the strongest biofilm inhibitory effect, while the addition of EDTA after 8 h had no biofilm inhibitory effects. EDTA was shown to inhibit cell-to-surface interactions and cell-to-cell interactions, which at least partially contributed to the repressed initial adherence. The addition of sufficient amounts of cations to saturate EDTA did not restore the biofilm formation, indicating the biofilm inhibition was not due to the chelating properties of EDTA. The study suggests that EDTA functions in the early stage of biofilm process by affecting the initial adherence of L. monocytogenes cells onto abiotic surfaces. Ó 2011 Elsevier Ltd. All rights reserved.

Keywords: Listeria monocytogenes Biofilm EDTA Initial adherence

1. Introduction Listeria monocytogenes is a Gram-positive food-borne pathogen which is responsible for listeriosis outbreaks (Kathariou, 2002). Listeriosis may have serious symptoms, including septicemia, abortion, liver failure, and meningitis, especially among susceptible people like immune-compromised individuals and pregnant women (Dussurget et al., 2004; Kathariou, 2002). This bacterium is ubiquitous in nature and can be found in many environments such as water, soil, sewage, silage and animal feces, where it is able to survive for several months or even years (Blackman and Frank, 1996; Kathariou, 2002). L. monocytogenes can stand a variety of environmental stresses. It has been reported that it can grow at refrigerated temperature, under high osmotic pressure (10% NaCl), and at a wide pH range (pH 4e9) (Cole et al., 1990). Biofilms are complex communities of microorganisms attached to surfaces and held together by extracellular polymeric substances (Davey and O’Toole, 2000; O’Toole et al., 2000). One of the major causes for concern about L. monocytogenes in food processing environments is its ability to attach to many different

* Corresponding author. Tel.: þ1 413 545 1016; fax: þ1 413 545 1262. . E-mail address: [email protected] (L. McLandsborough). 0740-0020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.07.009

surfaces and form biofilms (Borucki et al., 2003; Moltz and Martin, 2005; Moretro and Langsrud, 2004). Biofilms are much more resistant to detergents, biocides, and antibiotics than their nonattached, individual, free-living (planktonic) counterparts (Davey and O’Toole, 2000; McLandsborough et al., 2006; O’Toole et al., 2000). As a result, the biofilm-coated surfaces are particularly difficult to decontaminate, and conventional methods of disinfection or inactivation such as chemical oxidant and antibiotic treatments are often ineffective against biofilm bacteria (Davey and O’Toole, 2000; McLandsborough et al., 2006; O’Toole et al., 2000). EDTA (ethylenediametetraacetic acid) is a synthetic chelating agent that forms strong complexes with cations, and it thus has been widely used in food systems as a stabilizer and sequestrant (Winter, 1999). EDTA has also been shown to possess antimicrobial effects, by limiting the availability of essential cations for growth, and/or by destabilizing the cell membrane of bacteria by complexing divalent cations that act as salt bridges between membrane macromolecules, such as lipopolysaccharides (LPS) (Banin et al., 2006; Leive, 1965; Vaara, 1992). This vhas led to the use of EDTA as a preservative in many products. However, research about the influence of EDTA on bacterial biofilms is still limited, most of which focused on the use of EDTA as part of an antimicrobial catheter lock solution to prevent catheter-related infections caused

Y. Chang et al. / Food Microbiology 29 (2012) 10e17

by clinical microorganisms, including Staphylococcus, Pseudomonas, and Candida (Juda et al., 2008; Percival et al., 2005; Raad et al., 2003, 2008, 2007; Ramage et al., 2007; Yakandawala et al., 2007). Moreover, all of these researchers used high levels of EDTA, ranging from several times to several hundred times the minimal inhibitory concentration (MIC), either to inhibit biofilm formation by growth inhibition, or to eradicate mature biofilm by the bactericidal effect and/or chelating property of EDTA molecules. To our knowledge, the effect of EDTA on biofilm formation of L. monocytogenes has not been reported yet. Here we reported for the first time that the use of EDTA at low concentration levels significantly reduced the initial biofilm formation of L. monocytogenes. This effect is neither due to growth inhibition nor the chelating property of EDTA. This research suggests a potential use of EDTA, either alone or combined with other chemicals, to prevent biofilm formation of L. monocytogenes on food processing surfaces. 2. Materials and methods 2.1. Bacterial strains and media The strains used in this study were L. monocytogenes LM21 (Scott A) and 7 other strains (Table 1). The liquid medium used for growing L. monocytogenes was tryptic soy broth with 0.6% yeast extract (TSBYE), and the agar medium was TSBYE with 1.5% agar (TSAYE) (BD, Sparks, MD). Monthly working cultures were transferred from corresponding frozen culture to TSAYE slants, incubated at 32  C for 24 h and stored at 4  C. Prior to every experiment, a loop of strain was transferred from the working culture slant into 10 ml TSBYE that was incubated at 32  C stagnantly for about 18 h. All the biofilm assays were performed by incubating L. monocytogenes strains in a minimal defined media, Modified Welshimers Broth (MWB) (Premaratne et al., 1991), since MWB media has been shown to enhance biofilm formation of L. monocytogenes (Djordjevic et al., 2002). 2.2. Quantitative assay of biofilm formation Biofilm formation on polyvinyl chloride (PVC) was performed according to the methods previously described by Djordjevic et al. (2002) with minor modifications. Overnight cultures of L. monocytogenes in TSBYE were diluted 100-folds in MWB media. Inoculated MWB broth was then added to eight replicate wells of PVC microtiter plate (BD Falcon, Franklin Lakes, NJ) (100 ml/well) with the appropriate concentration of EDTA and incubated at 32  C for 48 h. After incubation the media were aspirated and microtiter

Table 1 L. monocytogenes strains used in this studya L. monocytogenes CU isolation no.b Lineage Persistencec Origin lab designation no. LM13 LM14 LM15 LM19 LM21 LM22 LM31 LM32

FSL-N1-025 FSL-N1-005 FSL-N1-332 FSL-N1-048 FSL-J1-225 (Scott A) FSL-J1-023 FSL-J2-020 FSL-J2-064

I II I I I III I I

NP NP P P

Fish processing plant Fish processing plant Fish processing plant Fish processing plant Human epidemic

Animal, cow Animal, cow

a All strains information was obtained from our previous publication (Djordjevic et al., 2002). b Strains were originally obtained from M. Wiedmann, Cornell University (CU). c P, persistent strain; NP, nonpersistent strain.

11

plate wells were washed 3 times with distilled water to remove loosely associated bacteria. Plates were air dried for 1 h at 56  C or overnight at room temperature, and each well was then stained with 150 ml of 0.1% crystal violet solution in water for 30 min. After staining, plates were washed 4 times with distilled water. At this point, biofilms were visible as purple rings formed on the side of each well. The quantitative analysis of biofilm production was performed by adding 190 ml of 95% ethanol to destain the wells for 1 h. A volume of 150 ml from each well was transferred to a new microtiter plate and the level of the crystal violet present in the destaining solution was measured at 570 nm using the microtiter plate reader (Bio-TEKÒ ELX800, Winooski, VT). 2.3. Assays of EDTA influence on biofilm formation Sterile disodium EDTA (Fisher Scientific, Fair Lawn, NJ) stock solution (10 mM EDTA, pH 8.0) was prepared for all the following experiments. EDTA was added to MWB media at varying levels (0.001 mMe0.3 mM, final concentrations) at the beginning of inoculation, to assess the influence of EDTA levels on the biofilm formation of L. monocytogenes. Also, inoculated MWB media was supplemented with 0.1 mM EDTA at different incubation time points, to assess the EDTA adding time on L. monocytogenes biofilm formation. A “stage shift” experiment was performed as well to further assess at which stage EDTA functions against the biofilm formation. Briefly, inoculated MWB media supplemented with or without 0.1 mM EDTA were added to the microtiter plate wells, followed by incubation at 32  C for 16 h (stage I). The planktonic cells in wells were then removed and the wells were washed twice to eliminate the remaining EDTA. Sterile MWB media with or without 0.1 mM EDTA were then placed, followed by a second stage of incubation for another 32 h (stage II). After the two stages incubation for a total of 48 h, the biofilms were then assayed. To assess whether Mg2þ or Fe3þ could overcome the inhibitory action of EDTA on biofilm production, an additional 0.2 mM MgCl2 or ferric citrate was pre-added into MWB media to saturate the subsequently added 0.1 mM EDTA, followed by inoculation and biofilm assay as previously described. To exclude the influence of the additionally added Mg2þ or Fe3þ on biofilm formation, corresponding controls were also conducted. To determine whether existing biofilms could be eliminated by the addition of EDTA, the L. monocytogenes strain LM21 was allowed to form biofilms on PVC microtiter plate wells for 48 h at 32  C in MWB in the absence of EDTA. After aspirating off the media and washing twice to remove loosely associated cells, PBS solution containing varying levels of EDTA (0, 0.1, 1, 10, 100 mM) was placed in the wells and incubated at 32  C for 24 h, to treat the established biofilms. After EDTA treatments for 24 h, the remaining biofilms were then quantified by crystal violet staining as described previously. All the above biofilm assays were performed three times using freshly prepared materials, and the averages and standard deviations were calculated accordingly for all repetitions of experiments. 2.4. Cellesurface interaction assay The cellesurface interaction assay was performed according to the method previously described (Shanks et al., 2008) with modifications. Overnight grown L. monocytogenes cultures were inoculated 1:100 into MWB media with or without 0.1 mM EDTA, and 30 ml of inoculated media were then added to 100  15 mm Petri dishes previously loaded with 12  50 mm PVC slides. After 4 h of adhesion at 32  C, PVC slides were taken out and nonadherent cells were gently washed out by PBS solution (pH 7.4).

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Differential interference contrast (DIC) microscopy was used to observe the adherent cells on the PVC slides. Digital images of several fields were taken and later counted for the numbers of adherent cells.

stabbing into two parallel motility MWA tubes, one with 0.1 mM EDTA and one without EDTA, followed by incubation at 32  C for 5 days. Inoculated tubes were inspected daily. Growth diffusing from the center stab was compared to check if the addition of EDTA affected the motility abilities.

2.5. Aggregation assay 2.9. Statistical analysis

2.6. Bacterial surface charge (z-potential) measurements The z-potential of L. monocytogenes cells treated with or without 0.1 mM EDTA was measured by a NanoZS ZEN3600 instrument (Malvern Instruments, Worcestershire, UK), using the method described by George and Kishen (2008) with modifications. Briefly, overnight grown L. monocytogenes cells were centrifuged and washed twice by sterile MWB media, and were then suspended in MWB supplemented with or without EDTA (0.1 mM). After treatment at 32  C for 2 h, L. monocytogenes cells were washed and suspended in appropriate amount of distilled water to achieve OD600 around 0.5. The z-potential of the bacterial suspension was then measured at room temperature. 2.7. Bacterial surface hydrophobicity measurements The surface hydrophobicity of L. monocytogenes cells treated with or without 0.1 mM EDTA was measured by a classic BATH (Bacterial adhesion to hydrocarbon) assay described by Rosenberg et al. (1980) with modifications. Briefly, freshly grown cells were washed and resuspended in appropriate amounts MWB to achieve an OD600 of 1.0 (0.1). EDTA solution was added to the suspensions to achieve the desired EDTA concentration (0.1 mM); for none EDTA-treated controls, the same volume sterile distilled water was added. After treatment at 32  C for 2 h, OD600 of the Listeria cell suspension was recorded (Ai). A volume of 1.5 ml cell suspension was then placed into a clean borosilicate glass tube (12  75 mm), followed by an addition of 1.5 ml of hexadecane (Sigma, purity  99%). The tube was vortexed for 2 min and set aside to rest for 15 min to allow the hexadecane phase to rise completely. Next, the cell suspension was retrieved with a clean Pasteur pipet, while taking great care to avoid taking in the hexadecane layer into the pipet. The cell suspension was then transferred to a cuvette for the final OD600 measurement (Af). Adhesion of cells to the hydrocarbons was evaluated as the fraction partitioned to the hydrocarbon phase, FPC, which could be calculated as FPC (%) ¼ 100 * (Ai  Af)/Ai. The value of FPC was used to evaluate the surface hydrophobicity of cells. 2.8. Motility test Soft motility agar (Modified Welshimer’s agar, MWA) tubes were prepared according to the method described by Djordjevic et al. (2002). The final agar concentration was 0.5% in 1 MWB with 0.1 mM EDTA or without EDTA (control). L. monocytogenes LM21 overnight growth in TSBYE 32  C was inoculated by needle

Microsoft Excel software was used to determine P values using the Student’s two-tailed pair-wise t test. Error bars are shown as one standard deviation. P < 0.05 was considered to be statistically significant. 3. Results 3.1. Low concentration of EDTA inhibited L. monocytogenes biofilm formation without affecting its planktonic growth The biofilm forming abilities of eight representative strains of L. monocytogenes were examined in the absence and presence of EDTA (0.1 mM), using the 96-wells microtiter plate assay previously described. The results were shown in Fig. 1 in which all strains exhibited significantly decreased biofilm formation with the addition of 0.1 mM EDTA (P < 0.05 for all the strains). This suggests that the biofilm inhibitory effect of EDTA is a general phenomenon for all tested strains of L. monocytogenes (Fig. 1). The L. monocytogenes strains LM21 and LM22 showed most biofilm reduction in reaction to EDTA; strain LM21 was selected for further study since this strain (Scott A) is frequently used in laboratory analysis. The influence of EDTA concentration against biofilm formation of the strain LM21 was tested over a range of 0e0.3 mM. The degree of biofilm inhibition by EDTA exhibited an obvious doseeffect correlation (Fig. 2). The biofilm levels were reduced in the presence of EDTA, even at the concentration as low as 0.001 mM (P < 0.05), with the biofilm reduction by 14.1%. The effect of biofilm inhibition gradually enhanced with the increased concentration of EDTA, reaching the highest biofilm repression at 0.3 mM EDTA (58.6% reduction) (P < 0.01), while 0.1 mM EDTA levels resulted in a 50.5% biofilm reduction (P < 0.01) (Fig. 2). Our preliminary observation showed that the addition of 0.3 mM EDTA

1.6 No EDTA

1.4

Biofilm Value (OD 570nm)

Aggregation assay was performed according to the method previously described by Shanks et al. (2008) with modifications. Briefly, aggregation of cells was determined by stagnantly growing 5 ml cultures in MWB with or without 0.1 mM EDTA at 32  C for 24 h in 16  150 mm test tubes. The upper 0.5 ml was carefully removed and the optical density (OD600) was measured (recorded as OD600 prevortex). The culture tube was then vortexed to resuspend aggregated cells, and 0.5 ml of this suspension was removed to measure its OD600 (recorded as OD600 postvortex). The “percent aggregation” was calculated using the formula: 100% * (OD600 postvortex  OD600 prevortex)/OD600 postvortex.

0.1 mM EDTA

1.2 1 0.8 0.6 0.4 0.2 0 LM13

LM14

LM15

LM19

LM21

LM22

LM31

LM32

Fig. 1. The inhibitory effect of EDTA against biofilm formation of eight representative strains of L. monocytogenes. EDTA (0.1 mM) was added to MWB media upon the inoculation of each strain. The biofilm was stained with crystal violet and detained with ethanol after 48 h incubation in PVC microtiter plates at 32  C. All strains exhibited significantly decreased biofilm formation with the addition of 0.1 mM EDTA (P < 0.05). The information of the strains was listed in Table 1.

Y. Chang et al. / Food Microbiology 29 (2012) 10e17

3.2. EDTA only affected the early stage of L. monocytogenes biofilm formation

80

60

Biofilm reduction (%)

13

40

20

0 0.001

0.01

0.1

1

EDTA concentration (mM) Fig. 2. Effect of EDTA concentration on biofilm formation of L. monocytogenes strain LM21. EDTA of various concentrations (as indicated) was added at the starting time of biofilm formation. Biofilm reduction is based upon the level of biofilm without the addition of EDTA. The biofilm reduction caused by varying levels of EDTA (0.01e0.3 mM) exhibited an obvious dose-effect correlation.

led to extended lag phase of LM21 growth (data not shown), while 0.1 mM EDTA did not (as shown in following). We therefore used 0.1 mM EDTA for all subsequent experiments to exclude EDTA’s potential growth-inhibitory effect. To further test whether biofilm inhibition by EDTA resulted from the growth-inhibitory effect, the growth of all eight cultures in MWB media with or without 0.1 mM EDTA were monitored using optical density measurements during microtiter plate incubation. The growth curves obtained showed that the addition of 0.1 mM EDTA had no effect on planktonic growth kinetics of each strain (Fig. 3, growth curves of strain LM21 as a representative), which demonstrated that the biofilm inhibition was not due to growth inhibition.

To determine at which stage EDTA influences biofilm production, 0.1 mM of EDTA was added at different time intervals during growth in microtiter plates. The results showed that adding EDTA at time zero had the most inhibitory effect against the biofilm formation, and the addition of EDTA after 4 h had much less effect (P < 0.05), while applying EDTA after 8 h or later did not show significantly inhibitory effect against biofilm formation (Fig. 4). We thus reason that EDTA only affected the early stage of L. monocytogenes biofilm formation. This hypothesis was further supported by a “stage shift” experiment (Fig. 5). The data showed that the presence of EDTA at the first incubation stage (0e16 h) inhibited biofilm formation, whether or not EDTA was present in the second incubation stage (16e48 h), indicating that the existence of EDTA during the first stage was sufficient to inhibit the biofilm formation. The addition of EDTA only in the second stage did not affect biofilm formation (Fig. 5), which was in accordance with the previous observation (Fig. 4). Together these data suggest that EDTA functions by inhibiting the early stages of L. monocytogenes cells to abiotic surfaces. 3.3. EDTA inhibited cell-to-surface interactions and cell-to-cell interactions during the biofilm formation of L. monocytogenes Because biofilm formation is dependent on both cell-to-surface interactions and cell-to-cell interactions (McLandsborough et al., 2006), we thus assessed the influence of EDTA on these two kinds of interactions. We first determined the effect of 0.1 mM EDTA upon cell-to-surface interactions by counting the cell numbers attached to PVC surface after 4 h adhesion using microscopy. Our data showed that EDTA-treated L. monocytogenes LM21 cells adhered in significantly lower numbers (P < 0.05) onto PVC surface (Fig. 6), indicating that EDTA inhibits cell-to-surface interactions. A dramatic representation of EDTA inhibition of cell-to-cell interactions was also observed when cultures were grown in MWB media in the absence versus in the presence of 0.1 mM EDTA

60 1

50

Biofilm reduction (%)

0.9

Optical density (OD570)

0.8 0.7 No EDTA 0.6

0.1 mM EDTA

0.5 0.4

40 30 20 10

0.3

0 0.2

-10

0.1

0

10

20

30

40

50

0 0

10

20

30

40

50

Time (hours) Fig. 3. Effect of EDTA (0.1 mM) on planktonic growth of L. monocytogenes strain LM21 in MWB media. The culture was grown in MWB without EDTA (dotted line) or with 0.1 mM EDTA (solid line) at 32  C in microtiter plates. Growth was monitored based upon optical density at 570 nm.

EDTA adding time (hours) Fig. 4. Effect of the time of EDTA addition on biofilm formation by L. monocytogenes strain LM21. EDTA (0. 1 mM) was added to microtiter plates at different time points (as indicated) during a total of 48 h biofilm incubation. Percent biofilm reduction is based upon the level of biofilm without the addition of EDTA. Biofilm reduction caused by EDTA decreased with the time of EDTA addition (before 8 h), and applying EDTA after 8 h or later had no inhibitory effect against biofilm formation.

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0.9

Biofilm Value (OD 570 nm)

0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 Stage I: 0.1 mM EDTA Stage II: 0.1 mM EDTA

Stage I: 0.1 mM EDTA Stage II: 0 mM EDTA

Stage I: 0 mM EDTA Stage II: 0.1 mM EDTA

Stage I: 0 mM EDTA Stage II: 0 mM EDTA

0

Fig. 5. Effect of EDTA presence at different incubation stages on biofilm formation by L. monocytogenes strain LM21. L. monocytogenes culture was inoculated in MWB with or without the presence of EDTA (0.1 mM) for the first stage of incubation for 16 h (stage I). At 16 h, the growth media was removed and replaced with fresh MWB with or without 0.1 mM EDTA, followed by a second stage of incubation for another 32 h (stage II). After incubation for a total of 48 h at 32  C, media were removed and biofilms were assayed.

(Fig. 7). Without the addition of EDTA, an aggregate of cells formed on the bottom of the test tube while the remaining culture was optically clear. In contrast, cultures incubated in the presence of EDTA grew as uniformly turbid cell suspensions. This effect was quantified, and the percent aggregation in the absence of EDTA was calculated as 90.3  12.2%; this value decreased to 32.5  3.6% when 0.1 mM EDTA was included in the culture media (P < 0.01). The formation of aggregation is an indication of stronger cellecell interactions (Shanks et al., 2008). The much less aggregation

formation upon addition of EDTA suggests that EDTA also inhibits the cellecell interactions. 3.4. EDTA inhibitory effect was not due to the depletion of Mg2þ or Fe3þ As EDTA chelates multivalent cations, it is reasonable to question whether the biofilm inhibition was due to EDTA chelating

1000

Number of cells / field

800

600

400

200

0 0 EDTA

0.1 mM EDTA

Fig. 6. Effect of EDTA (0.1 mM) on cell adhesion onto abiotic surfaces. L. monocytogenes LM21 cells adhered onto PVC surfaces by incubation for 4 h at 32  C in MWB media with or without 0.1 mM EDTA supplementation. The number of adherent cells per microscopic field was determined by DIC microscopy. EDTA-treated culture showed a significant decrease in the number of adherent cell numbers (P < 0.05).

Fig. 7. Visual aggregation assay with the culture (L. momocytogenes strain LM21) grown in the absence versus presence of EDTA. The culture was grown stagnantly for 24 h at 32  C in MWB media supplemented without EDTA (left) or with 0.1 mM EDTA (right).

Y. Chang et al. / Food Microbiology 29 (2012) 10e17

Flagella mediated motility was demonstrated to be critical for L. monocytogenes biofilm formation (Lemon et al., 2007), while L. monocytogenes cells were reported to lose flagella upon 0.3 mM EDTA treatments (Zaika and Fanelli, 2003), we therefore examined the influence of EDTA on the flagellar motility. The soft agar motility test revealed that addition of 0.1 mM EDTA did not influence the culture’s motility properties (data not shown). We also tested the effects of EDTA on the biofilm formation of a flagella minus mutant s19-5F (Chang and McLandsborough, unpublished data), and found that 0.1 mM EDTA further reduced its biofilm formation by 48.4  4.6% (P < 0.01), which is a similar reduction as observed with the wild type strain LM21 (Fig. 2). Together these data indicate that biofilm reduction in the presence of EDTA is not associated with flagellar motility.

1.2

Biofilm value (OD 570nm)

1.0

0.8

0.6

0.4

0.2

0.0 0E

DT

D 0E

A

TA

+0

.

M 2m

2 Mg

0E

+

DT

A

+

m 0 .2

M

Fe

3+ 0.

0.

M 1m

M 1m

ED

ED

TA

TA

.

M 2m

0.

M 1m

+0

2 Mg

ED

+

TA

+

m 0 .2

M

Fe

3+

3.6. EDTA did not destroy existing L. monocytogenes biofilms, even at high concentrations

Fig. 8. Effect of cations saturated EDTA on biofilm formation by L. monocytogenes strain LM21. EDTA (0.1 mM) was saturated by additional added multivalent cations (Mg2þ or Fe3þ, 0.2 mM) in MWB media. The same levels of Mg2þ or Fe3þ were added to MWB to run as controls.

existing cations in the media such as magnesium and iron. Considering that the only multivalent cations present in MWB media are Mg2þ (1.7 mM) and Fe3þ (0.36 mM), we therefore preadded additional 0.2 mM Mg2þ or Fe3þ to MWB media to saturate the subsequently added 0.1 mM EDTA, in order to determine whether the reduction in magnesium or iron was responsible for the decreased biofilm formation. Our results showed that the addition of 0.2 mM MgCl2 or ferric citrate did not overcome the inhibitory action of EDTA on biofilm production (Fig. 8), demonstrating that biofilm inhibition was not due to the depletion of cations such as Mg2þ and Fe3þ from growth media. 3.5. EDTA did not influence the L. monocytogenes cell surface properties (surface charge, surface hydrophobicity) or flagellar motility Cell surface properties such as surface charge and surface hydrophobicity are important factors for biofilm formation especially for the initial attachment step (McLandsborough et al., 2006), we thus examined the effect of EDTA on these factors. We compared the surface charge of L. monocytogenes LM21 cells treated with or without EDTA (0.1 mM) by measuring their respective zpotential, and no significant differences were observed (Table 2), indicating that EDTA did not have measurable effect on modifying the cell surface charge. Similarly, we also compared the surface hydrophobicity of LM21 cells treated with or without EDTA (0.1 mM) using BATH assay, and no significant differences were found either (Table 2), suggesting that EDTA at low levels did not influence the L. monocytogenes cell surface hydrophobicity.

Table 2 Measured surface charge (z-potential) and relative hydrophobicity L. monocytogenes strain LM21 treated without or with EDTA (0.1 mM). Treatments

Surface charge (z-potential, mV) Ave.  SD

Relative hydrophobicity (%)a Ave.  SD

No EDTA 0.1 mM EDTA

49.8  1.9 48.6  2.5

19.6  2.2 20.7  2.8

15

of

a BATH (bacterial adhesion to hydrocarbons) assay was used to determine the relative hydrophobicity. Hexadecane was the hydrocarbon used in this test.

EDTA could not disrupt the pre-existing L. monocytogenes biofilms, even at as high as 100 mM EDTA levels (data not shown). It could be possible that a portion or all the L. monocytogenes cells in the pre-formed biofilm were killed or damaged by the bactericidal effect of high dose of EDTA; however, the biofilm structures were not substantially destroyed as revealed by biofilm values quantified by the crystal violet staining. 4. Discussion The bacterial cells in biofilms are much more resistant to disinfectant and cleaning procedures. Consequently, the approaches to prevent biofilm formation or eradicating the formed biofilms are of special interest. In this study, we assessed the influence of EDTA on formation of L. monocytogenes biofilm on PVC material. This study was based upon an occasional laboratory observation which occurred when a typical DNA buffer (TriseEDTA, 1 mM : 0.1 mM, final concentration in media) was found to be inhibitory to biofilm formation by L. monocytogenes. We hence did further research and discovered that the inhibition was due to the presence of EDTA. In this study, we showed that low levels of EDTA are not inhibitory to planktonic growth of L. monocytogenes, but instead inhibit its biofilm formation by modifying the ability of L. monocytogenes cells adhering onto PVC surfaces. We have shown this decrease of biofilm occurs only if EDTA is present early in the biofilm formation process (Figs. 4 and 5). This is likely due to a decrease in initial adhesion, since on the presence of EDTA there appears to cause a significant decrease during initial cell attachment (Fig. 6) and has some effects upon the cell-to-cell aggregation (Fig. 7). As EDTA is a chelating agent, the presence of this chemical is likely binding multivalent cations such as magnesium and ferric iron which exist in MWB media. We initially hypothesized the biofilm inhibition was due to EDTA chelating. However, the addition of cations (Mg2þ or Fe3þ) to saturate EDTA did not restore the inhibitory effect of EDTA on biofilm formation (Fig. 8), indicating that the biofilm inhibition was not caused by the chelating properties of EDTA. The role of cell surface charge in the attachment of L. monocytogenes to surfaces was addressed previously (Chae et al., 2006; Dickson and Koohmaraie, 1989; Dickson and Siragusa, 1994) and we assumed that the cell surface would be more negatively charged by removal of cations from bacterial cells through the EDTA chelation, as observed by other researchers who used EDTA to treat Enterococcus faecalis cells (George and Kishen, 2008). Using zpotential measurement, a very sensitive method based upon

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cellular movement within an electric field, no differences were observed between EDTA treated and non-treated cells, therefore we do not believe the reduced biofilm production is due to changes in surface charge. Cellular hydrophobicity is another physiochemical factor which may influence bacterial adhesion on surfaces (Chae et al., 2006; Di Bonaventura et al., 2008; Takahashi et al., 2010). EDTA has been known to cause increased permeability of the outer membrane of Gram-negative bacteria (McDonnell and Russell, 1999), we therefore questioned if EDTA has the similar effect on L. monocytogenes cells and changes their cell surface hydrophobicity. In this study, the classic BATH method was used (Rosenberg et al., 1980) to measure the potential modification of cellular hydrophobicity. No differences in cell surface hydrophobicity were observed with cells treated with 0.1 mM EDTA and those not treated, indicating that it is unlikely that the reduced cellular adhesion and biofilm formation were due to measurable changes in cellular hydrophobicity. We also examined the influence of EDTA on flagellar motility of L. monocytogenes cells. However, based on results of the motility test and the fact that the biofilm formation of flagella minus mutant could be further reduced to the same levels as that of the wild type strain, it seems that EDTA affects biofilm formation independently of flagellar motility. The observed biofilm inhibition was not due to a pH effect, because the pH of MWB media supplemented with 0 and 0.1 mM EDTA were both 7.0, plus MWB itself has high buffering capacity (Premaratne et al., 1991). The addition of EDTA slightly diluted the MWB media. However, the biofilm inhibition was not due to the dilution of nutrients in MWB media either, since controls were performed by adding sterile water to MWB media to achieve the same degree dilution as the addition of EDTA, and the biofilm values were not changed (data not shown). It is unclear at this point how low concentration of EDTA causes this inhibition in biofilm formation, though the data presented here suggested that it is not by influencing motility, cell surface charge, hydrophobicity or depletion of metal ions. In summary, this study has presented, for the first time, that EDTA at sub-inhibitory concentration efficiently reduced biofilm formation of L. monocytogenes without affecting its planktonic growth. Adding EDTA at the starting time of biofilm formation had the strongest inhibitory effect against the biofilm formation, while the addition of EDTA after 8 h had no inhibitory effects. We also demonstrated that the existence of EDTA during the early stage was sufficient to inhibit biofilm formation, whether or not EDTA was present in the following stages. The study suggests that EDTA functions in the early stage by affecting the initial adherence of L. monocytogenes cells onto abiotic surfaces, possibly through the inhibition of cellesurface interactions and cellecell interactions. Although EDTA is a strong chelator of multivalent cations, the biofilm inhibitory effect does not seem to be caused by its chelating property. Our results indicate that EDTA acts either directly or indirectly to modify the way bacteria cells interact with surfaces, but obviously more work will be needed to elucidate the mechanisms further. Acknowledgments The authors thank Dr. John Cotter for his critical reading of the manuscript. This work was supported by the National Institute of Food and Agriculture, the Massachusetts Agricultural Experiment Station, U.S. Department of Agriculture, under the project number MAS00936 and Bioactive Foods Research for Health and Food Safety project MAS0201001529. This article is Paper Number 3471 of the Massachusetts Agricultural Experiment Station.

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