Food Protection Trends - March/April 2021 - 196

leading cause of economic concerns associated with the main
foodborne pathogens, causing an estimated economic burden
of $3.7 billion in a typical year in the United States (17)
and 32,900 disability-adjusted life years (27). In addition to
causing consumer insecurity, the aforementioned economic
burden, and public health challenges in the form of illness,
hospitalization, and death episodes, nontyphoidal Salmonella
serovars are listed in a recent report from the U.S. Centers
for Disease Control and Prevention (5) as a " serious threat "
for developing antibiotic resistance to medically important
classes of antibiotics.
As one of the main foodborne bacterial pathogens of
concern in infants, Cronobacter sakazakii similarly has been
associated with several national and international outbreaks
and is particularly lethal for neonates and preterm infants
(16). Since the first reported outbreak of C. sakazakii in
1958, infant formula has been associated with an array of
domestic and international episodes of infant morbidity
and mortality (16, 21).
More than six decades since the initial description of
microbial cells in sessile conditions and bacterial biofilms
(40), they still remain a major challenge in public health
microbiology, health care facilities, and food manufacturing
(9, 25, 30). Microbial biofilms are systematic congregations
of microorganisms confined to an extracellular autogenous
polymeric matrix, adhering to either biotic or abiotic surfaces
(7, 8, 15). The single or multiple species of microorganisms
present in biofilms are, in most cases, protected against
environmental stressors, including antimicrobials (18),
disinfectants and sanitizers, and immunity of the host (20).
This augmentation in resistance causes crucial challenges in
decontamination and treatment of microorganisms in health
care settings and production facilities (11).
Chlorine-based disinfectants including sodium hypochlorite are one of the most efficacious, cost-effective, and
commonly used sanitizing agents in domestic environments,
food processing facilities, and health care settings (24, 35).
Stainless steel is an ideal material for manufacturing surfaces
and machinery equipment due to its physicochemical stability
and high resistance to corrosion (19, 29). Nevertheless, a
myriad of examples are available in microbiology and medical
literature associated with biofilm formation of pathogenic
microorganisms on stainless steel surfaces (28, 29, 36).
An emerging technology in food manufacturing and the
pharmaceutical industry is the use of pressure-based pasteurization for ensuring the safety of an array of products (1-4, 22).
Although utilization of this emerging and efficacious nonthermal
technology is gaining momentum and rapid adaption in the
food manufacturing and pharmaceutical industries (2, 4), at
the current time very limited pieces of information are available regarding the fate and proliferation of pressure-stressed
pathogenic microorganisms, i.e., those that are exposed to sublethal hydrostatic pressure (23). For this reason, the purpose
of this study is to compare the biofilm formation capability

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Food Protection Trends March/April

of wild-type and pressure-stressed Salmonella serovars and
C. sakazakii at low and ambient temperatures on the surface
of stainless steel. The study also investigates the sensitivity of
both pathogens (Salmonella serovars and C. sakazakii) and the
two phenotypes (wild-type and pressure-stressed) to sodium
hypochlorite at various stages of biofilm formation. Currently,
Salmonella serovars and C. sakazakii are, respectively, the
leading causes of foodborne deaths in American adults (26)
and the dominant pathogenic challenge in the manufacturing
of infant formula (16).
The current study investigates the biofilm formation of
wild-type and pressure-stressed Salmonella serovars and
C. sakazakii for up to 3 weeks on the surface of stainless steel
coupons and further investigates the sensitivity of planktonic
cells and 1-week and 2-week-mature biofilms of both
phenotypes of the pathogens to sodium hypochlorite at 7 and
25°C. Note that the current study explores the sensitivity of
the pathogens to the widely used commercial sanitizer under
conditions that might not be easy to clean regularly, such
as niche and hard-to-reach manufacturing and processing
surfaces. Such areas could be a reservoir for mature bacterial
biofilms in processing areas.
MATERIALS AND METHODS
Bacterial culture preparation Strains of Salmonella serovars and C. sakazakii that were used in this study represent
two phenotypes of wild-type and pressure-stressed. The
cocktail of strains used in this study was selected based on
our preliminary work with individual strains (12) and on
epidemiological evidence for the selection of serovars with
public health importance (2, 3). Thus, a four-strain mixture
of Salmonella serovars (ATCC 13076 [Enteritidis], 8387
[Montevideo], 6962 [Newport], 14028 [Typhimurium])
and a four-strain mixture of C. sakazakii (ATCC BAA-894,
29544, 12868, 29004) were separately used in the current
study. The strains were activated, cultured, subcultured, and
purified individually and then were composited prior to
inoculation based on the methods articulated in our recent
open-access studies (1-4, 22). In short, for each strain, a
loopful from −40°C frozen glycerol and pathogen stock was
transferred into 10 mL of tryptic soy broth (TSB; Difco, BD,
Franklin Lakes, NJ) supplemented with 0.6% yeast extract
(YE) to enhance the recovery of injured cells. Inoculated
tubes were then incubated for 24 h at 37°C. A 100-μL aliquot
of this bacterial suspension was streak plated onto the surface
of tryptic soy agar (TSA) supplemented with 0.6% YE and
was incubated at 37°C for 24 h. These plates of pure cultures
were kept at 4°C and were used to prepare the bacterial
culture for trials.
Prior to the experiments, individual strains were activated
by culturing a single colony from the abovementioned plates
kept at 4°C into 10 mL of sterile TSB + YE, followed by
incubation at 37°C for 22 to 24 h. A 100-μL aliquot was then
subcultured into a different tube containing 10 mL of TSB

Food Protection Trends - March/April 2021

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