Original Article

Evaluation of the Removal and Destruction Effectof a Chlorine and Thiamine Dilaurylsulfate Combined

Treatment on L. monocytogenes Biofilm

Sokunrotanak Srey,1 Shin Young Park,1 Iqbal Kabir Jahid,1 Se-Ra Oh,1 Noori Han,1

Cheng-Yi Zhang,2 Soon-Han Kim,2 Joon-Il Cho,2 and Sang-Do Ha1

Abstract

The present study investigated the efficacy of single and combined treatment of both chlorine and thiaminedilaurylsulfate (TDS) on the reduction of Listeria monocytogenes biofilms in microtiter plate. The disinfectantsused in this study were 50, 100, and 200 mg/L chlorine and 100, 500, and 1000 mg/L of TDS. Biofilm-formingindex (BFI) and culturable cell count were used to evaluate the disinfectant assay. The highest BFI reductionwas 0.80, achieved by the combination of 200 mg/L chlorine and 1000 mg/L TDS. In contrast, the highestculturable cell count reduction was 4.80 log colony-forming units/well by the combination of 200 mg/L chlorineand 100 mg/L TDS. The BFI was reduced in a concentration-dependent manner while culturable cell count wassignificantly reduced only when all chlorine concentration was combined with 100 mg/L TDS. However, whenchlorine was combined with a higher concentration of TDS, the reduction decreased significantly. The result inthis study showed that the combination of the 200 mg/L chlorine and 1000 mg/L TDS could be a practicalapplication in removing L. monocytogenes biofilms from surfaces in food industry, and for the 200 mg/Lchlorine and 100 mg/L, it can be used for killing the pathogen biofilms. However, more studies are still neededin order to show its efficacy on foods surfaces as well as to develop an even more effective treatment in bothkilling and removing biofilms.

Introduction

L isteria monocytogenes is a psychrotrophic, Gram-positive, nonsporulating, and facultative anaerobe

foodborne pathogen, generally found in soil, water, sewage,vegetation, fecal matter, and animal feed (Moltz and Martin,2005). It is known as one of the common foodborne patho-genic bacteria that cause approximately 1600 illnesses eachyear in the United States, including more than 1400 hospi-talizations and 250 deaths (Scallan et al., 2011). It has beendocumented that L. monocytogenes is frequently found in rawmaterials and food-contact surfaces, so the environmentat the processing sites has often been examined in order toprevent the recontamination on the finished products (Kor-nacki and Gurtler, 2007). Furthermore, L. monocytogeneswas also found to be able to form biofilm (Chmielewski andFrank, 2006; Chaturongkasumrit et al., 2011). Recently, anoutbreak linked to whole cantaloupe contaminated withL. monocytogenes (CDC, 2011) was speculated to be causedby the pathogen firmly attached in an inaccessible place. It

can be said that those pathogens formed biofilms and theninternalized within the produce (Sapers, 2001). Biofilms havebeen recognized as a common source of chronic bacterial in-fection (Costerton et al., 1999). Biofilm formation comprisesbacteria that attach irreversibly to the surface and produceexopolymeric substances (EPS) (Agle, 2007). It is well docu-mented that biofilms have been playing an important role asa barrier for microorganisms against antimicrobial substances(Simoes et al., 2010). It is known that some strains ofL. monocytogenes can form biofilms and persist for years in thefood-processing environment (Wulff et al., 2006), and it wasalso reported that their biofilms are resistant to chlorine due tothe presence of EPS (Pan et al., 2006). Norwood and Gilmour(2000) reported that L. monocytogenes biofilm can evenwithstand 1000 mg/L sodium hypochlorite for 20 min. Sincethe current control strategies are not very effective, other newcontrol strategies have also been developed to counter thebiofilms problem such as enzyme (Lequette et al., 2010;Molobela et al., 2010), phage (Patel et al., 2011), and espe-cially hurdle technology (DeQueiroz and Day, 2007).

1School of Food Science and Technology, Chung-Ang University, Anseong-si, Gyeonggi-do, South Korea.2Ministry of Food and Drug Safety, Osong, Chungbuk, Korea.

FOODBORNE PATHOGENS AND DISEASEVolume 0, Number 0, 2014ª Mary Ann Liebert, Inc.DOI: 10.1089/fpd.2013.1734

1

NaOCl is a widely used chlorine-based disinfectant in thefood industry (Dychdala, 2001; Peng et al., 2002). It canionize via hydrolysis into HOCl, which is a strong bacteri-cidal agent (Fukuzaki, 2006). Additionally, thiamine dilaur-ylsulfate (TDS) is used as a synergistic agent with otherbactericides in noodles in Korea. Many authors have reportedits bactericidal activity and synergistic effect against food-borne pathogens (Kim et al., 2005; Lee et al., 2010; Kimet al., 2011), especially the combination of NaOCl and TDS(Lee et al., 2010; Kim et al., 2011). Studies concerningcombined treatment (hurdle technology) have shown re-markable improvement in reduction value. Lee and Ha(2008) demonstrated that combined treatment of disinfec-tants with TDS shows better reduction of bacteria coliformsin rice. However, there is no study on the effect of combinedtreatment of disinfectants with TDS on L. monocytogenesbiofilms yet. The purpose of this study was to evaluate theefficacy of combined treatment of sodium hypochlorite(NaOCl) with TDS on L. monocytogenes biofilm using abiofilm-forming index and culturable cell-count method.

Materials and Methods

Bacterial mix preparation

L. monocytogenes ATCC 19111 (serotype 1/2a, isolatedfrom poultry), ATCC 19113 (serotype 3b, isolated from hu-mans), and ATCC 19115 (serotype 4b, isolated from humans)were used as a mix in this study. The bacteria were recoveredfrom - 80�C frozen stock, and 100 lL of the stock was in-oculated into 10 mL of tryptic soy broth (TSB; Difco, Detroit,MD). Then, the solution was incubated at 30�C for 24 h. Aftervortexing, 100 lL of the incubated culture was pipetted into10 mL of fresh TSB followed by another 24-h incubation.The mix of the three strains was prepared with a 1:1:1 ratio.

Biofilm formation

Biofilms were formed in a microtiter plate following thepreviously described method by Stepanovic et al. (2004) andStepanovic et al. (2007) with some modifications. Twentymicroliters of the bacterial mix was inoculated into each wellof the 96-well flat-bottom microtiter plate (Becton DickinsonFalcon, Cockeysville, MD) followed by 230 lL of sterile TSB.The plate was sealed and statically incubated at 37�C for 48 h.According to preliminary experiment, which compared thebiofilm formation strength of different incubation tempera-tures and durations (data not shown), the strains used in thisstudy made strong biofilm formation at 37�C with 48-h incu-bation, so this condition was used in the biofilm formation forthe disinfection test. Two hundred fifty microliters of sterileTSB was used for negative control for the biofilm-formingindex (BFI) assay. BFI was calculated according to proceduresdescribed previously (Jahid et al., 2013). In biofilm formation,TSB was used instead of Brain Heart Infusion (BHI) broth,since TSB gave higher BFI than BHI (author’s observation inpreliminary experiment, data not shown).

Disinfectants preparation

Chlorine disinfectant solution was prepared using 12%NaOCl (6% free chlorine, Yakuri Pure Chemicals Co., Ltd.,Kyoto, Japan). The solution was diluted in sterile distilledwater to get a chlorine concentration of 50, 100, and 200 mg/L.

TDS, a vitamin B1 derivative, was first weighed and dis-solved in 30% ethanol solution to obtain a concentration of5%. Then, it was diluted in sterile distilled water or in theprepared chlorine solution to get a final solution with theconcentration of 100, 500, and 1000 mg/L. All disinfec-tants were prepared and used right after dilution duringdisinfection test.

Disinfection assay by BFI

After 48-h incubation, the optical density (OD) of the totalbacteria in the microtiter plate was measured at a wave-length of 600 nm (OD600nm; an optimum wavelength forestimating the concentration of bacteria; Matlock et al.,2011) with a microtiter plate reader (Spectra Max 190, Mo-lecular Devices, Sunnyvale, CA). The planktonic cells andmedium were removed, and each well was rinsed three timeswith 250 lL of sterile phosphate-buffered saline (PBS, pH7.2) to remove the loosely attached cells. Then, chlorine andTDS were added individually or in combination for 1 min;instead of disinfectant, PBS was used for treating positivecontrol (well with L. monocytogenes biofilm not subjected todisinfectant challenge) and negative control (well with TSBwithout L. monocytogenes biofilm). The disinfectant wasdiscarded by pipetting and 250 lL of Dey/Engley (D/E)neutralizing broth (Difco) was introduced into each well for5 min, including the control wells. Finally, the wells wererinsed three times with 250 lL PBS.

The biofilms were fixed with 250 lL of extra pure methylalcohol (Daejung Chemicals & Metals Co., Ltd., Shiheung,Korea) for 15 min; the plate was dried overnight in an up-inverse position after pouring out methyl alcohol. Crystalviolet dye 0.1% (CV; Sigma-Aldrich, St. Louis, MO) 250 lLwas used to stain biofilms, positive control, and negativecontrol wells for 15 min and removed by pipetting. Theplate was rinsed with tap water until the washing water wasdye free, and air-dried overnight. The bound dye was re-solubilized in 95% ethanol for 30 min and transferred into anew well plate. The OD of the dye solution was measured at570 nm (OD570nm).

The biofilm-removing efficacy of the disinfectants wascompared using the BFI calculated with the following for-mula (Niu and Gilbert, 2004):

BFI¼ (OD570nm�ODC570nm)

(OD600nm�ODC600nm)

where OD570nm is obtained from the disinfected (treated) orpositive control (biofilm treated with PBS) wells afterstaining and the ODC570nm is obtained from negative controlwells (TSB wells treated with PBS) after staining. TheOD600nm is obtained from the disinfected or positive controlwells, and ODC600nm is obtained from negative control wells,after 48-h biofilm formation. These OD values only re-presented the presence of cells that still attached to the sur-face after the treatment. It did not show the biological state ofL. monocytogenes.

Disinfection assay by culturable cell count

The biofilms were formed as stated previously. The testwas conducted following the method previously described(Teh et al., 2010) with some modification. After 48-h

2 SREY ET AL.

Table 1. Biofilm-Forming Index (BFI) and Culturable Cell Count of Listeria monocytogenes Biofilm

in Microtiter Plate After Challenging with Chlorine and Thiamine Dilaurylsulfate (TDS)

Concentration (mg/L) (log CFU/well)

Cl2 TDS BFI Reduction L. monocytogenes biofilm cells Reduction

0 0 1.33 – 0.04a — 8.17 – 0.06a —0 100 1.35 – 0.23a - 0.02 – 0.23f 8.17 – 0.08a 0.00 – 0.09l

0 500 0.89 – 0.04d 0.43 – 0.04cd 7.28 – 0.07c 0.89 – 0.07j

0 1000 0.77 – 0.02de 0.55 – 0.02bc 6.86 – 0.04d 1.30 – 0.04i

50 0 1.32 – 0.03a 0.01 – 0.03f 4.89 – 0.07g 3.27 – 0.07f

100 1.25 – 0.02bc 0.08 – 0.02f 4.74 – 0.05h 3.43 – 0.05e

500 0.89 – 0.03d 0.43 – 0.04cd 6.50 – 0.02e 1.66 – 0.02j

1000 0.65 – 0.04ef 0.67 – 0.04ab 7.09 – 0.07c 1.08 – 0.08k

100 0 1.07 – 0.02c 0.26 – 0.02e 4.21 – 0.02i 3.96 – 0.03d

100 1.04 – 0.06bc 0.28 – 0.06de 4.07 – 0.03j 4.10 – 0.03c

500 0.85 – 0.08d 0.47 – 0.08cd 4.74 – 0.06h 3.43 – 0.06e

1000 0.58 – 0.10f 0.75 – 0.09a 6.46 – 0.06e 1.71 – 0.07h

200 0 0.92 – 0.03cd 0.41 – 0.03cde 3.56 – 0.06k 4.61 – 0.06b

100 0.88 – 0.01d 0.44 – 0.01cd 3.37 – 0.01l 4.80 – 0.02a

500 0.83 – 0.01d 0.50 – 0.01c 4.12 – 0.01ij 4.04 – 0.01cd

1000 0.53 – 0.04f 0.80 – 0.04a 5.68 – 0.05f 2.48 – 0.05g

Mean – standard deviation values denoted by different superscript letters in the same column are significantly different ( p < 0.05).CFU, colony-forming unit.

FIG. 1. Field emission scanning electron microscopy images of Listeria monocytogenes biofilms in a microtiter plate. (A)Three-dimensional biofilm structure (arrows; 5000 · magnification). (B) Monolayer biofilms after treatment with 200 ppmchlorine and 1000 ppm thiamine dilaurylsulfate (5000 · magnification). (C) Exopolymeric substances remainder aftertreatment (circle; 20,000 · magnification). (D) Damaged cells remainder after treatment (arrow; 100,000 · magnification).

EFFECT OF CL/TDS ON L. MONOCYTOGENES BIOFILM 3

incubation, all planktonic cells and medium were removed andall wells were rinsed three times with 250 lL of PBS. Eachwell was treated with disinfectants as mentioned above. After1-min treatment, the disinfectant solutions were removed fromthe well and placed into 2 mL of D/E neutralizing broth (testtube A). Two hundred and fifty micro liters of D/E neutralizingbroth were introduced into each well. Then, a sterile cottonswab was pressed to the bottom of the well and rotated 50times clockwise and another 50 times anticlockwise. The swaband the remaining neutralizing solution in the well were alsoplaced into test tube A. After leaving for 5 min, the tubes werethen vortexed for 30 s each before serial dilution and spread-plating onto PALCAM Agar (Oxoid, Basingstoke, UK). Allplates were incubated at 30�C for 48 h prior to counting.

Field emission scanning electron microscopy (FESEM)

Processing of FESEM samples of biofilms was performedaccording to the procedures described previously ( Jahidet al., 2013) with some modifications. The biofilms devel-oped in a 12-well microtiter plates and after a disinfectanttest, biofilms were observed by using FESEM. Each well wasrinsed three times with PBS, pH 7.2. The adhered cells werefixed in 2% glutaraldehyde (Sigma Aldrich) in PBS for 24 hand then washed three times for 15 min with PBS. The fixedcells were serially treated with ethanol (50% for 15 min, 60%for 15 min, 70% for 15 min, 80% for 15 min, 90% for 15 min,and 100% two times for 15 min) and were successively de-hydrated by 33, 50, 66, and 100% hexamethyldisilazane(Sigma Aldrich) in ethanol for 15 min each. The dehydratedsamples were coated with platinum and observed usingFESEM. The FESEM was operated at an accelerating voltageof 5 kV and 5 mm working distance.

Statistical analysis

All treatments were conducted in three independent trials,with each having triplicate samples. Data were analyzed byone-way analysis of variance (Duncan’s test) using the Sta-tistical Analysis System (SAS; version 9.2, SAS InstituteInc., Cary, NC). Statistical significance was considered atp < 0.05.

Results and Discussion

Effect of combined treatment of chlorineand TDS on BFI of L. monocytogenes

It has been observed that BFI is a relatively new approach,and it is being used in biofilm research to identify biofilm-forming bacteria as well as to categorize bacteria biofilmformation strength (Niu and Gilbert, 2004; Naves et al., 2008;Moriyama et al., 2009; Teh et al., 2010). However, it is notcommonly used in the biofilms removal test even though itcan be considered a good means for biofilm quantification onthe surface, since biofilm is stained with crystal violet (CV),allowing the comparison of removal effect between thetreated and untreated biofilm. In the disinfectants or surfac-tants test, it can be postulated that BFI can be used as anindirect evaluation of the chemical efficacy in removingbiofilm from surfaces. This is because the BFI method allowsthe quantification of the amount of CV that is bound tothe remaining biofilm on the surface after biofilms havebeen challenged with disinfectants or surfactants. In this

experiment, 100 and 200 mg/L chlorine, and 500 and1000 mg/L TDS as single treatment showed significant( p < 0.05) removal of L. monocytogenes biofilm (Table 1). At500 and 1000 mg/L, TDS removed 0.43 (z32%), and 0.55(z41%) BFI, respectively; whereas 100 mg/L TDS showedlittle or no effect at all. It can be seen that the removal efficacyof chlorine or TDS was increased when they were mixed to-gether. All chlorine concentrations, when used in combinationwith 1000 mg/L TDS, showed good biofilm-removal proper-ties, which achieved a 0.67–0.80 BFI reduction, depending onthe chlorine concentration. These values were significantlyhigher than those of chlorine-only treatment. The highest re-moval was by the combination of 200 mg/L chlorine with1000 mg/L TDS, yielding 0.8 (z60%) BFI reduction. SinceTDS showed high BFI reduction, it can be speculated that TDSmight hold an interfacial tension-lowering property, leading toremoval of bacteria biofilm. TDS was also considered as asurfactant in another study (Fransisca et al., 2012).

Effect of combined treatment of chlorine and TDSon culturable cell count of L. monocytogenes

However, the disinfectants affect culturable biofilm cellsdifferently. As shown by the results, chlorine 50 mg/L,100 mg/L, and 200 mg/L could significantly ( p < 0.05) inac-tivate the cells by 3.27, 3.96, and 4.61 log colony-formingunits (CFU)/well, respectively (Table 2). Up to now therehave been no studies on the effect of chlorine and TDS onL. monocytogenes biofilm in microtiter plates; however,Ronner and Wong (1993) reported that chlorine 100 mg/Lcan reduce 4 log CFU/cm2 of L. monocytogenes biofilms onstainless steel surfaces after 2-min treatment. Mustapha andLiewen (1989) also observed a more than 4 log CFU/mLreduction of Listeria biofilm on stainless steel surfaces whentreated with 200 mg/L more than 2 min. These results couldbe speculated to be comparable to that of this study. On the

Table 2. Culturable Cells Count of Listeria

monocytogenes Biofilm After Challenging

with Chlorine and Thiamine Dilaurylsulfate (TDS)

(log CFU/well)

DisinfectantsConcentration

(ppm)L. monocytogenes

biofilm cells Reduction

Control — 8.17 – 0.06a —TDS 100 8.17 – 0.08a 0.00 – 0.09l

500 7.28 – 0.07c 0.89 – 0.07j

1000 6.86 – 0.04d 1.30 – 0.04i

Cl2 + TDS 50 0 4.89 – 0.07g 3.27 – 0.07f

100 4.74 – 0.05h 3.43 – 0.05e

500 6.50 – 0.02e 1.66 – 0.02j

1000 7.09 – 0.07c 1.08 – 0.08k

100 0 4.21 – 0.02i 3.96 – 0.03d

100 4.07 – 0.03j 4.10 – 0.03c

500 4.74 – 0.06h 3.43 – 0.06e

1000 6.46 – 0.06e 1.71 – 0.07h

200 0 3.56 – 0.06k 4.61 – 0.06b

100 3.37 – 0.01l 4.80 – 0.02a

500 4.12 – 0.01ij 4.04 – 0.01cd

1000 5.68 – 0.05f 2.48 – 0.05g

Mean – standard deviation values denoted by different superscriptletters are significantly different ( p < 0.05).

CFU, colony-forming unit.

4 SREY ET AL.

other hand, the results indicated that TDS alone is much lesseffective than treatment with only chlorine; the highest con-centration (1000 mg/L) of TDS only reduced the cell count by1.55 log CFU/well. Unexpectedly, the combined treatmentreduced the chlorine effect in a TDS concentration-dependentmanner. As illustrated in Table 2, the reduction was increasedto 3.43–4.80 log CFU/well when chlorine was combined with100 mg/L TDS. However, the reduction decreased significantlyto 1.66–4.04 log CFU/well when chlorine was combined with500 mg/L TDS. For the combination with 1000 mg/L TDS, thereduction decreased dramatically to 1.08–2.48 log CFU/well.The most effective combination for the reduction is 200 mg/Lchlorine and 100 mg/L TDS. The results showed that chlorineand TDS might have an antagonistic effect in killing L.monocytogenes biofilm (The effect of combined treatment wasless than the sum of the individual effect of TDS and chlorine)in a concentration-dependent manner since the reduction de-creased as the concentration of TDS increased. There was noreport on the effect of chlorine and TDS combined treatment onL. monocytogenes biofilm. These results were not consistentwith other research in which the two compounds showed asynergistic effect (i.e., higher synergism with higher concen-tration) on the bacteria reduction in foods (Lee et al., 2010; Haet al., 2012; Park et al., 2012). However, TDS is a derivative ofvitamin B1, and it has been documented that vitamin B1 isdestroyed by NaOCl (Dwivedi and Arnold, 1973; Bates, 2007);it is also unstable at neutral pH or can be destroyed at pH above8 (oxidation) (Bates, 2007). This could be a possible explana-tion that could potentially explain the decrease in the reductionvalue of culturable cell counts in combined treatment.

The difference between BFI and culturablecell count method

It should be noted that the BFI method and the culturablecell count method could provide two completely differentresults. The BFI method allows an estimation of the cells thatattach to a surface. It does not provide information on thebiological state of the cells. Thus, the results from BFI rep-resent only the cells remaining on the surface after thetreatment including the dead, culturable, and viable butnonculturable cells. This method allows us to indirectlyevaluate the removal effect of the disinfectant on the biofilm.

However, the culturable cell count method, used in thisstudy, represents all the culturable cells after the treatmentincluding the cells that were detached from the surface. Thus,this method provides the evaluation of the bactericidal effectof the disinfectants on the biofilm.

FESEM of L. monocytogenes treatedwith combined chlorine and TDS

The FESEM image showed that three-dimensional biofilmsof L. monocytogenes were formed after incubation at 37�C for48 h (Fig. 1A). After challenging with the combined treatment,the biofilms were removed and reduced to monolayer biofilms(Fig. 1B). The image showed that even though some cells wereremoved, the EPS still presented on the surface (Fig. 1C) aswell as the damaged cells (Fig. 1D). It has been reported that ifthe removal of bacteria does not provide a bactericidal effect,the microorganism will be able to accumulate elsewhere in theindustrial settings and reproduce biofilm (Gram et al., 2007).

It is also suggested that because the bacteria biofilm is killedbut not removed from the surface, it might act as an enhancerfor bacteria attachment since pre-existing EPS will facilitatethe adhesion (Flemming and Schaule, 1988; Tang et al., 2009).Therefore, it is necessary for researchers to study both theremoval and bactericidal efficacy of disinfectant on biofilms.

Conclusions

In conclusion, the combination of 200 mg/L chlorine and100 mg/L TDS achieved the highest culturable cell count re-duction, when used to treat L. monocytogenes biofilms. How-ever, the increase in reduction is relatively small (4.80 log CFU/well compared to single chlorine treatment 4.61 log CFU/well).The combination of 200 mg/L chlorine and 1000 mg/L TDSachieved the highest BFI reduction. It may be a practical ap-plication for biofilm removal (BFI reduction). Furthermore,future research should look into genes expression responding tochemical treatment in order to achieve a good synergism incombined treatment. In case of surface cleaning, both removaland bactericidal effect of the treatment should be evaluated,since it is important that the pathogens be removed and killed toprevent further contamination and biofilm formation.

Acknowledgments

This research was supported by funding from the 2012grant (12162KFDA012) from the Korea Food and DrugAdministration for studies on hazardous microbes and mi-crobiological safety management of seafood.

Disclosure Statement

No competing financial interests exist.

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Sang-Do Ha, PhDSchool of Food Science and Technology

Chung-Ang University72-1 Nae-Ri

Daedeok-Myeon, Anseong-siGyeonggi-do 456-756, Korea

E-mail: sangdoha@cau.ac.kr

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