Food Protection Trends - July/August 2022 - 279

attachment to the surface; (ii) aggregation into microcolonies in
a semipermanent association; and (iii) growth and maturation
of macrocolonies. At the end of the development stage, cells
are irreversibly attached to the surface. The microorganisms are
in a semipermanent association with the surface because the
embedded cells in the matrix have transitioned from a motile to
a sessile lifestyle. The biofilm is now mature, and its cells cannot
be removed by simply rinsing (1, 10, 32). Biofilms offer greater
protection to microorganisms against external challenges such
as temperature, pH, and antimicrobial solutions through the
secretion of extracellular polymeric substances (10). Factors
such as biofilm age and presence of other species or other L.
monocytogenes strains allow biofilms to acquire higher resistance
to sanitation strategies such as chemical sanitizers (4, 21). Such
resistance of mature biofilms is due to the protection provided
by the extracellular polymeric substances and the multiple layers
of bacterial cells in the biofilm (38).
Food processing facilities follow sanitation standard operating
procedures to ensure sanitary conditions in the processing plant
(24). The use of chemical sanitizers is one of the most common
practices for combating contamination, bacterial adhesion,
and biofilm formation (29). However, novel technologies such
as the use of UV light are being implemented to enhance the
effectiveness of standard sanitation methods and ensure the
safety of the products (18). Among the most commonly used
sanitizers are quaternary ammonium compounds (QACs),
organic acids, and peroxyacetic acid, and their antimicrobial
activity against biofilms have been previously investigated
(5, 8). When biofilms of L. monocytogenes were treated with
chemical sanitizer, reductions of 1 to 3 log CFU/cm2
were
observed (4, 21). However, results indicate that the use of
chemical sanitizer as a single strategy is not always sufficient
to control biofilms in food processing environments (4, 21,
26). Therefore, some sanitation standard operating procedures
include use of sanitizers in rotation or the implementation of
hurdle technologies (i.e., combined intervention strategies)
to achieve a greater antimicrobial effect. Among alternative
germicidal technologies, nonionizing UV irradiation has been
effective against bacterial biofilms (15, 19, 31). The combination
of UV light with chemical sanitizers also has been investigated.
A UV-C light lamp was ceiling mounted in a fish smokehouse
after daily cleaning and disinfection procedures, and a significant
decrease in Listeria-positive environmental samples was
reported after irradiation for 48 h (7). The combination of
sodium hypochlorite and UV light was used effectively against
L. monocytogenes biofilms in industrial kitchens, facilities, and
restaurants (19). Combined physical and chemical techniques
appear to represent a good strategy for overwhelming bacterial
responses and achieving a greater reduction of L. monocytogenes
biofilms.
The objectives of this study were to (i) investigate L. monocytogenes
biofilm survival after treatments with chemical
sanitizers (i.e., quaternary ammonium, lactic acid, and peroxy
acid) and UV-C light (254 nm) alone or in combination and
(ii) understand the effect of changing the sequence of treatments
to identify possible enhancement of overall antimicrobial
activity.
MATERIALS AND METHODS
Bacterial strains
The same strains of L. monocytogenes investigated in
previous research (25) were used for this study. FSL B2-323
(serotype 4b; Texas Tech University) was isolated from a dairy
processing environment (6), ATCC 7644 (serotype 1/2c)
was from a clinical case of human meningitis, NRRL B-33043
(serotype 1/2a; California) and NRRL B-33260 (serotype
1/2c; U.S. Department of Agriculture, Agricultural Research
Service) were obtained from a meat slaughter facility (36).
Each strain was kept in a CryoCare organism preservative
system (Key Scientific, Stamford, TX) and stored at −80°C
until needed for the experiments.
Biofilm formation
A biofilm-growing device developed by the Centers for
Disease Control and Prevention (https://biofilms.biz/
products/biofilm-reactors/cdc-biofilm-reactor/) was used
to develop 4-day-old biofilms on circular stainless steel
coupons 1.27 cm in diameter. A protocol developed in our
laboratory was used (25), and coupons were treated and
cleaned following the manufacturer's instructions. Each L.
monocytogenes strain was grown overnight at 37 ± 2°C in 10 ml
of tryptic soy broth (Difco, BD, Sparks, MD) with 0.6% yeast
extract (Hardy Diagnostics, Santa Monica, CA) (TSBYE).
Equal amounts of bacteria were combined to create a cocktail,
and 1 ml was used to inoculate the reactor containing 350 ml of
TSBYE. The initial cocktail population was verified by spread
plating on tryptic soy agar (TSA; Difco, BD) and enumerating
after 24 h of incubation at 37 ± 2°C. Biofilms were grown at 30
± 2°C following a 24-h batch phase and a 72-h continuous flow
stirring phase (11 ml/min flow rate and 60 rpm stirring force).
Chemical sanitizers exposure
Coupons with mature biofilms were removed from the
reactor after 4 days. Three chemical sanitizers were used at
room temperature for an exposure time of 10 min: 4% lactic
acid solution, pH 3 (Purac, Corbion, Blair, NE); a peroxy
acid-based sanitizer diluted to 100 ppm, pH 4.5 (SYNERGEX,
Ecolab, St. Paul, MN); and a quaternary ammonium-based
sanitizer (STER-BAC, Ecolab) diluted to 400 ppm, pH 6,
following the manufacturer's recommendations. Immediately
after treatment, solutions were neutralized by transferring the
coupons into 10 ml of D/E neutralizer broth (Difco, BD).
UV-C light treatments
Experiments to evaluate the effect of UV-C light on biofilms
were conducted in a small chamber in which UV irradiance
could be measured and controlled. UV-C light (254 nm) was
applied for 15 min (0.2 J/cm2
) or 30 min (0.45 J/cm2
) at room
July/August Food Protection Trends 279
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