Activity of disinfectants against foodborne pathogens in suspension and adhered to stainless steel surfaces

The purpose of this study was to investigate and compare the efficacy of various disinfectants on planktonic cells and biofilm cells of Listeria monocytogenes, Staphylococcus aureus and Escherichia coli. Numbers of viable biofilm cells decreased after treatment with all tested disinfectants (iodine, biguanide, quaternary ammonium compounds, peracetic acid and sodium hypochlorite). Sodium hypochlorite was the most effective disinfectant against biofilm cells, while biguanide was the least effective. Scanning electron microscopy observations revealed that cells adhered on stainless steel surface after treatment with the disinfectants. No viable planktonic cells were observed after treatment with the same disinfectants. Based on our findings, we concluded that biofilm cells might be more resistant to disinfectants than plancktonic cells.


INTRODUCTION
Microorganisms were shown to form biofilms on the surface of materials commonly used in food processing, such as stainless steel (3, 31); thus, these surfaces become potential source of contamination that may lead to food spoilage, transmission of diseases (19, 27, 31), equipment damage and compromise the sanitation of food surfaces and environmental surfaces by spreading detached organisms to other areas of processing plants (29). Biofilm formation may be separated into the primary attachment of bacteria to surfaces, followed by proliferation of the attached bacterial cells, which leads to the accumulation of multilayered clusters of cells and extracellular polymer (glycocalyx) formation (6, 10). Biofilm bacteria can be physically and morphologically different from their planktonic counterparts, especially in response to sanitizers and biocides (8,17,20,29,31).
It is well documented that bacteria, including foodborne pathogens such as Listeria monocytogenes, Staphylococcus aureus and Escherichia coli, can 'stick' to a variety of surfaces found in food industries (5, 14, 19, 24 28). To reduce or eliminate microorganisms on food contact surfaces, food processors have relied on techniques that have been proven over many years of use. These techniques include physical methods (e.g. hand washing, high pressure sprays) and chemical methods (e.g. hypochlorites, iodophores, quaternary ammonium compounds). Both techniques should remove and inactivate microorganisms that might be on the surface of equipment which may eventually come in contact with raw and processed food (4, 5, 14).

Preparation of suspension
The bacterial strains were grown overnight (18 to 24h) at 37ºC with shaking (150 revolutions per minute-rpm) in tryptic soy broth (DIFCO). Cells were harvested by centrifugation at 5,000 x g for 3.5 min and washed three times in phosphatebuffered saline (PBS; 0.1M, pH 7.2). Cell pellets were resuspended in PBS and adjusted by a spectrophotometer to an A660 of approximately 0.5, corresponding to ~ 10 8 CFU/ml (21).

Disinfectants
The disinfectants used in this study were chosen to represent those used in the food industry. The following disinfectants were used: iodine (0.20% w/v), biguanide (0.50% w/v), quaternary ammonium compounds (0.50% w/v), peracetic acid (0.50% w/v) and sodium hypochlorite (1.50% w/v). All the disinfectants used were provided by Johnson-Diverseylever, Brazil. These agents were diluted with sterilized distilled water according to the manufacturer's instructions.

Test surface
AISI type 304 stainless steel was the surface chosen as it is used extensively throughout the food processing industry.
Flat, stainless steel coupons (1 x 1cm) were used as the test surface to examine biofilm formation in vitro. The coupons were initially soaked overnight in acetone to remove grease.
After soaking, the steel coupons were placed in a sterile tube and sonicated for 15 min in a bath sonicator. The coupons were then washed in tap water followed by three washes with distilled water, and they were autoclaved at 121ºC for 15 min (23). The manipulations of coupons were assisted with a sterile surgical clamp for all assays.

Biofilm formation in vitro
A 20μl aliquot of the 10 8 CFU/mL suspension prepared as described above was placed in 50 mL plypropylene tubes (9) containing 15 mL of inoculated Mueller Hinton Broth (11) and one sterilized stainless steel coupon, and incubated at 37ºC under constant agitation of 100 rpm for 5 days. The culture medium was changed every 3 days. Biofilm formation was confirmed using scanning electron microscopy (25).

Activities of the tested disinfectants against the biofilm cells
After biofilm formation, coupons were rinsed twice with 5 mL of sterile physiological saline to remove any attached bacterial cells, and separately placed in Petri dishes containing 20 mL of one of the tested disinfectants at 25±2ºC for 10 min.
A positive control was performed by placing a coupon in a Petri dish containing 20 mL of sterile physiological saline. The coupons were removed from the dishes and immediately transferred to 5 mL of Letheen Broth (11) for 10 min to inactive the disinfectants and rinsed twice again with 5 mL of sterile physiological saline. They were then placed in glass tubes containing 5 mL of sterile physiological saline, sonicated Disinfectants against foodborne pathogens at 40kHz for 8 min (or 36kHz for 10 min) and vortexed for 10s (21). This procedure releases viable bacteria adhering to the coupons into the physiological saline. To quantify viable cells, bacteria were resuspended, serially diluted 10-fold with sterilized physiological saline and cultured in trypticase soy agar at 37ºC for 24-48h (21).
The experiment was repeated three times for each strain, and the mean and standard deviation were calculated.

Activities of the tested disinfectants against the planktonic cells
A 0.05 mL aliquot of 10 8 CFU/mL suspension prepared as described above was added to glass tubes containing separately 4.95 mL of each tested disinfectant at 25ºC±2ºC and vortexed for about 10s. A positive control was performed by adding a 0.05 mL aliquot of the suspension (10 8 CFU/mL) to a glass tube containing 4.95 mL of sterile physiological saline. From each tube, 0.5 mL was sampled after 10 min, added to 4.5 mL of Letheen Broth (11) to inactivate the disinfectants for 10 min, and vortexed again for 10s. Viable cells were counted as described above. The experiment was performed three times for each strain, and the mean and standard deviation were calculated (21).

Scanning electron microscopy observations
After treatment with the tested disinfectants, the stainless steel coupons were immersed in 2.5% glutaraldehyde in 0.

Statistical analysis
Numbers of CFU/cm 2 and CFU/mL were transformed to log 10 . The data were analyzed by analysis of variance (ANOVA) and the Tukey multiple comparison test using MINITAB Statistical Software (version 13.1). Statistical significance was defined as p<0.05.

RESULTS AND DISCUSSION
Poor sanitation of food contact surfaces, equipment, and processing environments has been a contributing factor in foodborne disease outbreaks (5, 8). In this study, we have demonstrated the efficacy of disinfectants used in food industries against foodborne pathogens in suspension and in biofilm. Table 1 shows the activities of the tested disinfectants on biofilm cells of S. aureus, L. monocytogenes and E. coli. The number of viable cells is presented as a logarithm. Statistically significant differences (p < 0.05) were found on average count of viable cells of all studied strains after treatment with the disinfectants.      It may be difficult to compare results from these different studies because the conditions for attachment and biofilm development vary greatly and these differences can be significant (5).
Once the microorganisms have attached, they must be capable of withstanding normal disinfection processes. Biofilm bacteria display a resistance to biocides that may be considered stunning (15).
According to Characklis and Marshall (7), incomplete removal of the biofilm will allow it to quickly return to its equilibrium state, causing a rebound in total place counts following sanitization. Surviving organisms rapidly create more extracellular polymers as a protective response to irritation by chemical cleaning agents.
It has become clear that biofilm-grown cells express properties distinct from planktonic cells, one of which is an increased resistance to antimicrobial agents (17). Studies have indicated that slow growth and/or induction of an rpo S -mediated stress response could contribute to biocide resistance (13,16). (1)    All the content of the journal, except where otherwise noted, is licensed under a Creative Commons License