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Applied and Environmental Microbiology, June 2009, p. 4089-4092, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02807-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Effect of Octenidine Hydrochloride on Planktonic Cells and Biofilms of Listeria monocytogenes

Mary Anne Roshni Amalaradjou,¹ Carol E. Norris,² and Kumar Venkitanarayanan^1*

Department of Animal Science,¹ Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269²

Received 10 December 2008/ Accepted 4 April 2009

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Listeria monocytogenes is a food-borne pathogen capable of forming biofilms and persisting in food processing environments for extended periods of time, thereby potentially contaminating foods. The efficacy of octenidine hydrochloride (OH) for inactivating planktonic cells and preformed biofilms of L. monocytogenes was investigated at 37, 21, 8, and 4°C in the presence and absence of organic matter (rehydrated nonfat dry milk). OH rapidly killed planktonic cells and biofilms of L. monocytogenes at all four temperatures. Moreover, OH was equally effective in killing L. monocytogenes biofilms on polystyrene and stainless steel matrices in the presence and absence of organic matter. The results underscore OH's ability to prevent establishment of L. monocytogenes biofilms by rapidly killing planktonic cells and to eliminate preformed biofilms, thus suggesting that it could be used as a disinfectant to prevent L. monocytogenes from persisting in food processing environments.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Listeria monocytogenes is a major bacterial pathogen (2), accounting for approximately 28% of the deaths resulting from food-borne illnesses in the United States (22). It is widespread in nature and occurs in soil, vegetation, fecal matter, sewage, water, and animal feed (14). Because it is ubiquitous, L. monocytogenes is frequently isolated from foods and food processing environments (13, 23), thereby presenting a significant challenge to the food industry. Several studies have shown that L. monocytogenes is capable of adhering to food contact surfaces, such as glass, stainless steel, rubber, and polystyrene (6, 11, 28). Upon attachment to such surfaces, L. monocytogenes establishes biofilms and persists for long periods of time in the food processing environment (18, 30). This potentially poses a food safety hazard since biofilms are an important source of contamination of food products that come into contact with them. In addition, biofilms also protect the underlying bacteria from desiccation, antimicrobials, and sanitizing agents (7, 16). Thus, eradication of L. monocytogenes biofilms in processing plants is critical for improving food safety.

When problems with persistent L. monocytogenes are encountered in food processing facilities, plant hygiene and sanitation are emphasized (31). This involves preventing the establishment of L. monocytogenes biofilms in the food processing environment and reducing contamination of product contact surfaces. A variety of cleaners and disinfectants, including quaternary ammonium compounds and hypochlorite, have been evaluated for this purpose (20). Although these compounds are approved by the Food and Drug Administration for use as disinfectants in processing plants, they are not effective in killing L. monocytogenes (24, 25), especially in the presence of soil or organic matter and at low temperatures. Therefore, there is a need for an effective disinfectant that can eliminate listerial biofilms in the presence of organic matter at a wide range of temperatures. Octenidine hydrochloride (OH) is a positively charged bispyridinamine that exhibits antimicrobial activity against plaque-producing organisms, such as Streptococcus mutans and Streptococcus sanguis (5). Toxicity studies with a variety of species have shown that OH is not absorbed through mucous membranes and the gastrointestinal tract, and there have been no reports of carcinogenicity, genotoxicity, or mutagenicity of this compound (17, 19, 29).

The objective of this study was to investigate the efficacy of OH for inactivating planktonic cells and preformed biofilms of L. monocytogenes at 37, 21, 8, and 4°C in the presence and absence of organic matter on two matrices, polystyrene and stainless steel.

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Culture preparation.
All bacteriological media were purchased from Difco (Becton Dickinson, Sparks, MD). The following three strains of L. monocytogenes were used in this study: 316 (pork), ATCC 19115 (human), and Scott A (human). Stock cultures were stored at –80°C in tryptic soy broth containing 0.6% yeast extract (TSBYE) and 5% glycerol. Working cultures were maintained on slants of tryptic soy agar containing 0.6% yeast extract (TSAYE) at 4°C. Prior to each experiment, a loopful of culture was grown in 10 ml of TSBYE incubated at 32°C for 24 h. The antimicrobial efficacy of OH for L. monocytogenes planktonic and biofilm cells was tested using all three strains individually.

Inactivation of planktonic L. monocytogenes.
Briefly, the L. monocytogenes strains were grown separately overnight in TSBYE at 32°C. Following incubation, the cultures were sedimented by centrifugation (3,600 x g for 15 min), washed twice with phosphate-buffered saline (PBS) (pH 7.2), and resuspended in 10 ml of TSBYE. Two hundred microliters of each washed culture was used as an inoculum (6.0 log CFU). Sterile 96-well polystyrene tissue culture plates (Falcon, Franklin Lakes, NJ) were inoculated with 200 µl of the cell suspension, which was followed by addition of 0, 1 (0.5 µl), or 5 (2.5 µl) mM OH (>99% pure; Dishman USA, Middlesex, NJ). The plates were incubated at 37, 21, 8, or 4°C. At zero time and following 1, 2, and 5 min of OH exposure, the surviving L. monocytogenes populations were enumerated by serial dilution (1:10 in PBS) and plating on duplicate TSAYE plates. The plates were incubated at 37°C for 24 h before the colonies were counted. When L. monocytogenes was not detected by direct plating, samples were tested for surviving cells by enrichment for 24 h at 37°C in 100 ml of TSBYE, followed by streaking on Oxford agar. Representative colonies on TSAYE were confirmed to be L. monocytogenes colonies based on colony morphology when samples were streaked on Oxford agar plates. Triplicate samples were used for each treatment, and the experiment was replicated three times.

Inactivation of biofilms formed on polystyrene.
The antibiofilm effect of OH was determined by a microtiter plate assay (12). Briefly, the L. monocytogenes strains were separately grown overnight in TSBYE, sedimented, washed as described above, and diluted 1:40 in TSBYE. Sterile 96-well polystyrene tissue culture plates (Costar, Corning Incorporated, Corning, NY) were inoculated with 200 µl of each cell suspension (6.0 log CFU) and incubated at 37 or 21°C for 24 h or at 8 or 4°C for 7 days without agitation. Following biofilm formation, the effect of OH was tested using 0, 10 (5 µl), and 20 (10 µl) mM OH and exposure times of 0, 2, 5, and 10 min. After exposure to OH for these times, the wells were washed three times with 200 µl of sterile PBS, dried at room temperature, and finally stained with 1% crystal violet for 15 min. After three rinses with sterile distilled water and subsequent destaining with 95% ethanol, the absorbance at 570 nm of each adherent biofilm was measured with a microplate reader (model 550; Bio-Rad, Hercules, CA). Uninoculated wells containing TSBYE were used as blanks. Blank-corrected absorbance values were used to express biofilm production. Five replicate wells were used for each treatment, and the assay was repeated three times.

Organic matter contamination.
The efficacy of OH for inactivating planktonic cells and biofilms of L. monocytogenes in the presence of organic matter was determined by using the method of Fatemi and Frank (15). Sterile rehydrated nonfat dry milk (100 g milk solids/liter) was used to simulate organic matter contamination in food processing plants. To determine the effect of OH on L. monocytogenes planktonic cells in the presence of organic matter contamination, nonfat dry milk was added to each treatment, which was followed by addition of OH (0, 1, or 5 mM) and incubation for 0, 1, 2, and 5 min at 37, 21, 8, or 4°C. Following exposure to OH, the surviving population was enumerated by viable plate counting on TSAYE plates, as described previously. Three samples were used for each treatment, and the assay was replicated three times. To determine the antibiofilm effect of OH in the presence of organic matter, listerial biofilms were grown in the presence of rehydrated milk and exposed to OH (0, 10, and 20 mM) for 0, 2, 5, and 10 min. The biofilms were assayed by colorimetry, as described above. This study was done using temperatures of 37, 21, 8, and 4°C. Five replicate wells were used for each treatment, and the assay was repeated three times.

Enumeration of bacteria in biofilms.
In addition to the microtiter plate assay, the antibiofilm effect of OH was assayed by enumerating surviving bacteria in biofilms using the viable plate count method (26). Following exposure to OH, the wells were washed three times with PBS, and the adherent biofilm was scraped off and plated directly or after serial dilution in PBS onto TSAYE plates. The plates were incubated at 37°C for 24 h before the bacterial colonies were enumerated. When L. monocytogenes was not detected by direct plating, samples were tested for surviving cells by enrichment for 24 h at 37°C in 100 ml of TSBYE, followed by streaking on Oxford agar.

Biofilm assay with a stainless steel matrix. (i) Preparation of stainless steel coupons.
Stainless steel (type 304 with a 4b finish) was used to make coupons (4). The stainless steel coupons (diameter, 1 cm) were washed and cleaned prior to use, as described by Djordjevic et al. (12). To remove grease, the coupons were soaked in acetone for 10 min and washed with distilled water. The coupons were cleaned with ethanol and rinsed thoroughly with distilled water. Then the coupons were boiled in distilled water for 10 min. In the final step, the coupons were autoclaved for 15 min at 121°C.

(ii) Biofilm assay.
L. monocytogenes cells were grown and diluted 1:40 as described above. Two hundred microliters of the inoculum was then dispensed onto the stainless steel coupons submerged in a 24-well plate (Falcon, Becton Dickson Labware, Franklin Lakes, NJ). Biofilms were formed at 37, 21, 8, or 4°C and treated with 0, 10, or 20 mM OH for 0, 2, 5, or 10 min. A procedure described by Ayebah and coworkers (4) was used to remove, disperse, and enumerate the cells in biofilms. At each sampling time, to enumerate the surviving L. monocytogenes cells, stainless steel coupons were placed in sterile Nalgene bottles (8 oz) containing 30 ml of sterile PBS and 3 g of acid-washed glass beads (425 to 600 µm; Sigma-Aldrich Co., St. Louis, MO). The bottles were shaken for 10 min on an orbital incubator at 400 rpm to dislodge the bacteria from the coupons. Serial dilutions of the PBS were prepared after this shaking and surface plated onto TSAYE. The plates were incubated for 24 h at 37°C before the colonies were counted. When L. monocytogenes was not detected by direct plating, samples were tested to determine the presence of surviving cells by enrichment for 24 h at 37°C in 100 ml of TSBYE, followed by streaking on Oxford agar. The biofilm assay with a stainless steel matrix was also performed in the presence of organic matter, as described above. Duplicate coupons were used for each treatment, and the experiment was replicated three times.

Confocal microscopy.
To obtain depth-selective information on the three-dimensional structure of the biofilms, in situ confocal laser scanning microscopy was performed. For microscopic assessment, biofilms were grown at 37°C in TSBYE using a Lab-Tech eight-chamber no. 1 borosilicate glass coverslip system (Lab-Tek, Nalge Nunc International, Rochester, NY). Microscopy was performed as described by Chae and Schraft (10). The biofilms formed on coverslips were treated with OH, and the live and dead cells were imaged after the preparations were stained with 2.5 µM SYTO (Molecular Probes, Oregon) and 5 µM propidium iodide (Molecular Probes, Oregon). Biofilms not exposed to OH (control) were also imaged to determine the normal architecture of L. monocytogenes biofilms. Samples were examined with a Leica true confocal scanner SP2 microscope using a water immersion lens. A krypton-argon mixed-gas laser with a PMT2 filter was used as the excitation source. For each sample, four randomly selected fields of view were analyzed. For each field, a series of images were taken in the z direction, beginning at the outer surface of the biofilm and continuing at 0.2-µm intervals.

Statistical analysis.
The data were analyzed using the Proc Mixed procedure of SAS (27). Statistical comparisons between different treatments were performed using Fisher's least significant difference. Differences were considered significant if the P value was <0.05.

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OH was found to be equally effective in inactivating the three strains of L. monocytogenes used in this study. Therefore, only the results obtained with ATCC 19115 are presented here. OH was effective not only in killing planktonic cells and preventing listerial biofilm formation but also in inactivating preformed L. monocytogenes biofilms. L. monocytogenes planktonic cells were rapidly inactivated (exposure time, 10 s) by 5 mM OH. A lower concentration, 1 mM OH, reduced the bacterial counts by more than 3.0 log CFU/ml on contact (Fig. 1). As expected, L. monocytogenes populations in control samples remained the same throughout the sampling period. It was also observed that the antimicrobial effect of OH was not affected by temperature. OH at concentrations of 1 and 5 mM resulted in similar reductions in pathogen population at 37°C (Fig. 1), 21°C, 8°C, and 4°C. When OH was tested using planktonic cells of L. monocytogenes in the presence of rehydrated milk, it retained its antimicrobial efficacy, and the killing was similar to that observed in the absence of organic matter (data not shown). OH also exhibited a significant antibiofilm effect (P < 0.05) against L. monocytogenes at all four temperatures. For biofilms on both polystyrene and stainless steel, 20 and 10 mM OH completely inactivated the biofilms (negative as determined by enrichment) after 10 s and 5 min of exposure, respectively (Fig. 2 and Fig. 3). Like the activity against planktonic cells, the activity of OH against biofilms was not affected by the incubation temperature or the presence of organic matter (data not shown). To investigate the effect of OH on biofilm structure, L. monocytogenes biofilms that formed on glass coverslips were analyzed by confocal microscopy. Positive staining using SYTO and propidium iodide was used for imaging. The confocal images of the control biofilm (Fig. 4A) with no added OH revealed the formation of a dense biofilm (average thickness, 14 µm; maximum thickness, 20 µm) containing green cells (live) stained by the SYTO dye, while the images of the treated samples (Fig. 4B) revealed patchy breaks in the biofilms due to loss of cells and disruption of organization, as indicated by red cells (dead) stained by propidium iodide. The average thickness of OH-treated biofilms was 1 µm, and the maximum thickness was 3 µm.

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FIG. 1. Inactivation of L. monocytogenes planktonic cells by OH at 37°C. Sterile 96-well polystyrene tissue culture plates were inoculated with 200 µl of a cell suspension, which was followed by addition of 0, 1 or 5, mM OH. The plates were incubated at 37°C. At zero time and following 1, 2, and 5 min of exposure to OH, L. monocytogenes populations were enumerated. No significant effect of temperature on the antimicrobial effect of OH was found.

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FIG. 2. Inactivation of L. monocytogenes biofilms on polystyrene by OH at 37°C. Sterile 96-well polystyrene tissue culture plates were inoculated with 200 µl of a cell suspension (6.0 log CFU) and incubated at 37°C for 24 h without agitation. Following biofilm formation, the effect of OH was tested using concentrations of 0, 10, and 20 mM and exposure times of 0, 2, 5, and 10 min. Colorimetry was performed to assess the antibiofilm effect of OH. No significant effect of temperature on the antibiofilm effect of OH was found.

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FIG. 3. Inactivation of L. monocytogenes biofilms on stainless steel by OH at 37°C. Two hundred microliters of inoculum was dispensed onto stainless steel coupons submerged in a 24-well plate. Biofilms were formed at 37°C and treated with 0, 10, or 20 mM OH for 0, 2, 5, or 10 min. Following exposure, the surviving cells in the biofilms were dispersed and enumerated. No significant effect of temperature on the antibiofilm effect of OH was found.

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FIG. 4. Confocal microscopy of L. monocytogenes biofilms without OH treatment (A) and after treatment with OH (B). L. monocytogenes biofilms formed on coverslips were treated with OH, and the live and dead cells were imaged after staining with 2.5 µM SYTO and 5 µM propidium iodide. Samples were examined with a Leica true confocal scanner SP2 microscope using a water immersion lens.

The ability of OH to retain its efficacy against biofilms in the presence of organic matter is significant for the food industry since several studies have shown that the commonly used disinfectants have reduced efficacy in the presence of organic matter (3, 8). OH also exhibited the same efficacy for killing planktonic cells and biofilms at all temperatures tested, whereas hypochlorite, quaternary ammonium compounds, and iodophors have reduced efficiencies for killing biofilms at temperatures below room temperature (21). This is important because the internal temperatures of food processing plants are sometimes maintained at temperatures below room temperature. OH exerts its antimicrobial effect by binding to the negatively charged bacterial cell envelope, thereby disrupting the vital functions of the cell membrane and killing the cell (9). It has high affinity for cardiolipin, a prominent lipid in bacterial cell membranes, making it selectively lethal to bacterial cells without adversely affecting eukaryotic cells (9). In addition, Al-Doori and coworkers (1) reported that repeated exposure of Staphylococcus aureus to OH for up to 3 months did not induce resistance to this compound, suggesting that regular use of OH as a disinfectant may not induce resistance in L. monocytogenes.

In conclusion, OH effectively killed planktonic cells of L. monocytogenes and rapidly inactivated preformed biofilms on both polystyrene and stainless steel. In addition, OH was equally effective against listerial biofilms at different temperatures and in the presence of organic matter. These results indicate that OH can be used as a disinfectant or sanitizer in food processing plants to prevent and inactivate L. monocytogenes biofilms. However, this hypothesis needs to be confirmed by field trials.

 
* Corresponding author. Mailing address: Department of Animal Science, Unit 4040, 3636 Horsebarn Hill Road Extension, Storrs, CT 06269. Phone: (860) 486-0947. Fax: (860) 486-4375. E-mail: Kumar.venkitanarayanan{at}uconn.edu

Published ahead of print on 17 April 2009.

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Applied and Environmental Microbiology, June 2009, p. 4089-4092, Vol. 75, No. 12
0099-2240/09/$08.00+0     doi:10.1128/AEM.02807-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Amalaradjou, M. A. R. Articles by Venkitanarayanan, K. Search for Related Content PubMed PubMed Citation Articles by Amalaradjou, M. A. R. Articles by Venkitanarayanan, K. Agricola Articles by Amalaradjou, M. A. R. Articles by Venkitanarayanan, K.