Disinfection Treatment of Heated Scallop-Shell Powder on Biofilm of Escherichia coli ATCC 25922 Surrogated for E. coli O157:H7

Miki Kubo, Yori Ohshima, Fumio Irie, Mikio Kikuchi, Jun Sawai

Faculty of Applied Bioscience, Kanagawa Institute of Kanagawa, Atsugi, Japan.

DOI: 10.4236/jbnb.2013.44A002                

Abstract

The ability of heated scallop-shell powder (HSSP) to disinfect Escherichia coli ATCC 25922 biofilm was investigated. On account of its cryotolerance and cell surface characteristics, the E. coli strain is reportedly a useful surrogate for E. coli O157: H7 in surface attachment studies. In this study, an E. coli ATCC 25922 biofilm was formed on a glass plate, and immersed in a slurry of HSSP. Following treatment, the disinfection ability of the HSSP toward the biofilm was non-destructively and quantitatively measured by conductimetric assay. The disinfection efficacy increased with HSSP concentration and treatment time. HSSP treatment (10 mg/mL, pH 12.5) for 20 min completely eliminated biofilm bioactivity (approximately 10⁸ CFU/cm² in non-treated biofilms). In contrast, treatment with NaOH solution at the same pH, and treatment with sodium hypochlorite (200 mg/mL) reduced the activity by approximately one to three log₁₀. Fluorescence microscopy confirmed that no viable cells remained on the plate following HSSP treatment (10 mg/mL). Although alkaline and sodium hypochlorite treatments removed cells from the biofilm, under these treatments, many viable cells remained on the plate. To elucidate the mechanism of HSSP activity against E. coli ATCC 25922, the active oxygen generated from the HSSP slurry was examined by chemiluminescence analysis. The luminescence intensity increased with increasing concentration of HSSP slurry. The results suggested that, besides being alkaline, HSSP generates active oxygen species with sporicidal activity. Thus, HSSP treatment could also be effective for controlling biofilms of the toxic strain E. coli O157: H7, implicated in food poisoning.

Keywords

Scallop-Shell Powder; Biofilm; Escherichia coli; Conductimetric Assay; Fluorescence Microscopy; Active Oxygen; Alkali

1. Introduction

Bacteria can become tenaciously attached to food and its contact surfaces by forming biofilms [1]. Biofilms are more resistant to environmental stresses such as nutritional and oxidative stresses, desiccation, UV light exposure, and sanitizing agents, than free microorganisms [2]. Biofilms attached to food contact surfaces such as stainless steel, polyvinyl chloride and polyurethane are a continuous source of food spoilage bacteria and pathogens in food processing environments [3-6]. Among the biofilm-forming species is the enterohemorrhagic strain Escherichia coli O157:H7 [7]. Enterohemorrhagic E. coli strains produce Shiga-like toxins, and E. coli O157:H7 infection is potentially fatal, particularly in young children and the elderly [8,9].

Previous studies [10,11] have demonstrated the strong antibacterial activity of heated scallop-shell powder (HSSP). The main component of scallop-shells is CaCO₃, which, when heated, generates the powerful bactericidal agent CaO [12]. In fact, the antibacterial activity of powder heated to 1000˚C is comparable to that of pure CaO [10]. HSSP treatment effectively reduces the aerobic bacterial count in shredded cabbage [13] and kills Bacillus subtilis spores [14,15]. We have previously investigated the disinfection efficacy of HSSP against biofilms of Salmonella sp. [16] and Staphylococcus aureus [17]. HSSP treatment (10 mg/mL, pH 12.5) completely eliminated bioactivity of the biofilm (approximately 10⁷ CFU/cm² in non-treated biofilms) and was more effective than or nearly as effective as sodium hypochlorite (200 ppm = 200 mg/L) against Salmonella sp. and S. aureus, respectively. In contrast, treatment with NaOH was less effective than HSSP slurry at the same pH. Bodur and Cagri-Mehmetoglu [18] also reported that HSSP treatment removed biofilms of three pathogens, Listeria monocytogenes, S. aureus and E. coli O157:H7, formed on stainless steel plates.

In the present study, we investigated the disinfection efficacy of HSSP against biofilms of E. coli ATCC 25922 on glass plates. This E. coli strain is reportedly a useful surrogate for E. coli O157: H7 in surface attachment studies, since it is cryotolerant and possesses desirable cell surface characteristics [19]. The cell populations of apples contaminated with E. coli ATCC 25922 and E. coli O157: H7 were similarly reduced after washing the apples with hydrogen peroxide [20]. The two strains are similarly resistant to alkaline pH and heat [21] and similarly sensitive to gamma radiation in meats [22] and UV pasteurization in apple cider [23]. In the present study, biofilms grown on glass plates were subjected to HSSP treatment, and the metabolic and growth activities of the cells were determined by conductimetric assay, without disrupting the biofilm structure. The viability of the E. coli ATCC 25922 cells remaining on the plate after HSSP treatment was determined by fluorescence microscopy. In addition, to clarify the mechanisms of disinfectant efficacy, the active oxygen generated from the HSSP slurry was investigated by chemiluminescence analysis.

2. Materials and Methods

2.1. Preparation of Sample Slide Glass Plate of Biofilm

Escherichia coli ATCC 25922(=NBRC15034) was purchased from the National Institute of Technology and Evaluation Biological Resource Center (NRBC). The bacteria were stored in 10% glycerol solution at −85˚C. Prior to experiment, they were thawed and pre-incubated in nutrient broth (Eiken Chemicals Co. Ltd., Tokyo, Japan) at 37˚C for 24 h. The pre-incubated E. coli ATCC 25922 cells were washed and resuspended in 10 mL sterile saline. A portion (0.1 mL) of the bacterial suspension was added to a 100-mL plastic vial (IWAKI, Asahi Glass Co. Ltd.) containing 40 mL of Todd-Hewitt Broth (Oxoid, Cambridge, UK). A sterilized slide glass plate (76 × 26 mm, Matsunami Glass Ind. Ltd., Tokyo, Japan) was immersed in the broth and incubated at 37˚C for 48 h (110 strokes/min). Following incubation, the plate was removed from the broth and gently washed twice with 10 mL sterile deionized water (hereafter, this plate is called the BF plate). For the conductimetric assay, a small piece (10 mm × 26 mm) of the prepared biofilm plate was cut with a diamond cutter.

2.2. HSSP Treatment

Powder from scallop-shells (Patinopecten yessoensis) was obtained from Soycom Co. Ltd. (Atsugi, Kanagawa, Japan). The powder was heated at 1000˚C in air for 1 h and ground in a ball mill. The approximate mean size of the HSSP particles was 5 µm. The calcium and magnesium content of scallop shell powder heated at 1000˚C for 1 h was 70.8 wt% and 0.16 wt%, respectively. Trace amounts of phosphorus (0.073 wt%), sodium (0.014 wt%), and iron (0.003 wt%) were also present. Silver and copper, which possess antibiotic properties, were not detected (<0.01 mg/kg). At a calcium concentration of 70.8 wt%, the shell contains 99% CaO by weight (Sawai et al., 2001). The powder was mixed with sterile saline (0.85 w/v%) to produce a slurry. The prepared biofilm plate was immersed in a vial containing the prepared HSSP slurry (100 mL) and incubated at 37˚C (110 strokes/min).

The above procedure was repeated for sodium hydroxide (NaOH) and sodium hypochlorite (Wako Pure Chemical Industries) treatments. NaOH and sodium hypochlorite were solubilized in sterile deionized water.

2.3. Evaluation of Biofilm Activity by Conductimetric Assay

Electric conductivity was measured by a RABIT™ (Rapid Automated Bacterial Impedance Technique) system (Don Whitley Scientific Ltd., UK). Conductivity was measured in a capped sample tube containing paired electrodes at the bottom. Conductimetric assays detect microbial growth and metabolism as changes in the electrical conductivity or impedance of the growth medium [24]. Such conductivity changes indicate the presence of electrolytes produced by metabolic activity, such as the conversion of glucose to lactic acid and/or increased mobility caused by the cleavage of large charged molecules (such as proteins) into smaller, more mobile charged molecules (such as amino acids). When the difference between two successive electrical conductivity measurements of the medium exceeds 5 µS, the RABIT™ system detects the change. The time required to reach the threshold concentration is called the TTD (time to detection); the TTD values may be used as criteria for determining bacterial growth [24].

The BF plates exposed to HSSP or other treatments were gently washed twice with 10 mL sterile deionized water, and immersed in the RABIT sample tubes containing 5 mL Whitley Impedance Broth (WIB: Don Whitley Scientific Ltd.). An untreated BF plate was used as a control. The tubes were set in the incubator module, and the conductivity changes in the growth medium, induced by metabolism and growth of E. coli, were monitored at 37˚C at 6 min intervals for 48 h.

Growth of E. coli ATCC 25922 was calibrated from the planktonic cells and the cells attached to the glass plate. To assess planktonic cell growth, serial ten-fold dilutions of E. coli suspensions were prepared with sterile saline. A 0.1 mL aliquot of each diluted suspension was added to the RABIT sample tube containing 5 mL WIB, and the conductivity change was measured at 37˚C for 48 h at 6 min intervals.

For assessing the growth of cells attached to the glass plate, serial ten-fold dilutions of suspended cells were also prepared with sterile saline. A 5 mL aliquot of E. coli suspension was spread onto the glass plate (10 mm × 26 mm) and dried. The dried plate was immersed in the RABIT sample tube containing 5 mL of WIB, and the conductivity change was measured at 37˚C for 48 h at 6 min intervals.

2.4. Fluorescence Microscopic Investigation of BF Treated with HSSP

The BF plates exposed to HSSP and other treatments were stained with LIVE/DEAD BacLight™ Bacterial Viability Kit (Invitrogen Co., CA, USA). This assay uses SYTO9 and propidium iodide (PI) to discriminate between live cells with intact membranes (green fluorescence) and dead cells with compromised membranes (red fluorescence). The cells were stained according to the manufacturer’s instructions. The stained BF plate was examined by fluorescence microscopy (Olympus FV1000D IX81, Tokyo, Japan).

2.5. Chemiluminescence Analysis of Active Oxygen Species

Active oxygen species generated from the HSSP react with luminol and trigger a chemiluminescent response, as described by Kohtani et al. [25]. A Mithras LB09470 microplate reader (Berthold Technologies, Bad Wildbad, Germany) was used for measurements. Samples of HSSP slurry (100 μL) were pipetted into 96-well microplates, and the chemiluminescence reaction was initiated by adding 50 μL of 0.7 mM luminol (Nacalai Tesque, Kyoto, Japan). Chemiluminescence was then recorded by the microplate reader. To examine the effects of antioxidative enzymes, 25 µL of 0.1 mg/mL superoxide-dismutase (SOD) or 0.1 mg/mL catalase solution (Wako Pure Chemical Industries) was added to the wells from the dispenser prior to luminol addition.

Each experiment was performed in triplicate and the average values are reported in the results.

3. Results

Figure 1 plots the typical conductivity curves for E. coli ATCC 29522 biofilm treated with 0.1mg/mL HSSP. The ordinate is the conductivity change (%) in the WIB growth medium. The conductivity of the untreated biofilm plate became detectable at approximately 4.5 h (i.e. TTD = 4.5 h). TTD was delayed at longer treatment times, being 10.7 h and 11.3 h after treatment for 20 min and 40 min, respectively. No TTD was detected after 60-min treatment.

From the TTD values obtained in the conductimetric assay, the number of remaining viable cells in the HSSP-treated biofilm was estimated. Figure 2 plots the calibration curves of planktonic cells and plate-attached cells of E. coli ATCC 29522. The ordinate is the initial

Figure 1. Typical conductivity curves for E. coli ATCC 25922 biofilms treated with HSSP at 0.1 mg/mL (pH = 11.6). ○: control, ●: 20 min, ▲: 40 min, ■: 60 min. Data points with bars represent means ± standard error.

Figure 2. Calibration curves of E. coli ATCC 25922; planktonic cells (○) and cells attached to the plate (●) in conductimetric assays. Data points with bars represent means ± standard error.

number of E. coli cells inoculated in the RABIT sample tube, obtained by the plate count method. For cells in both states, the TTD shortened as the initial number of inoculated cells increased. However, at a given TTD value, the populations of E. coli cells in the biofilm estimated from the planktonic-cell calibration curve were underestimated by two or three orders of magnitude. Since the number of viable attached cells better matched the proliferation values for biofilms in the RABIT tube than the number of viable planktonic cells, the viable cell counts in HSSP-treated biofilms were estimated from the attached-cell calibration curve (the conversion equation is: viable cell counts [log CFU/tube] = −0.38 × TTD + 10.7; R² = 0.97).

The viable counts estimated from the calibration curve of the attached cells are summarized in Table 1. The estimated viable cell count in the control biofilm was 8.1 log CFU/cm². HSSP treatment (0.1 mg/mL) for 20 and 40 min reduced the viable count by two log₁₀. No conductivity changes occurred after HSSP treatment at 0.1 mg/mL for 60 min, indicating that the E. coli cells in the biofilm were completely dead. Increasing the HSSP concentration to 1.0 mg/mL caused an approximately 2.5-order reduction in viable cell count after 40 min, and no TTD was observed at longer treatment times. At 10 mg/mL, HSSP treatment for 20 min completely suppressed activity (i.e. no TTD was detected). In contrast, NaOH treatment at pH 12.5 (the pH of 10 mg/mL HSSP slurry) reduced the viable cell count by one to three orders of magnitude. Although no TTD was detected after 60 min treatment with 200 ppm sodium hypochlorite, sodium hypochlorite was less effective than HSSP at both concentrations.

Figure 3(a) is a fluorescence microscope image of untreated E. coli biofilm grown on a glass plate (control). The biofilm is layered with intact cells (green) and dead

Conflicts of Interest

The authors declare no conflicts of interest.

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