Filter sterilized carcass rinsate for recovery of Salmonella species with various concentrations of cetylpyridinium chloride

Abstract Controlling Salmonella in poultry processing continues to be important to processors and consumers. Cetylpyridinium chloride (CPC) has proven to be effective in vitro in controlling Salmonella. This study evaluated the recovery of Salmonella after overnight storage in 4°C filter‐sterilized carcass rinsate containing CPC from 0.44 to 909 ppm (μg/mL). Ten Salmonella serotypes (18 strains), of which 6 serotypes are commonly isolated from poultry products, were grown in Bacto‐Tryptic Soy Broth overnight at 37°C. Serial dilutions of a CPC/propylene glycol solution were prepared in 24‐well tissue culture plates containing filter‐sterilized carcass rinsate. Approximately 107 cfu/mL of each Salmonella serotype was added to the appropriate wells. Inoculated plates were stored overnight at 4°C. After storage, triplicate plates of brilliant green agar with sulfapyridine (BGS) were surface inoculated with 10 μL of the contents for each well, streaked for isolation, and incubated at 37°C for 24 h. Three replications were conducted. The presence of typical colonies on BGS plates was recorded as growth and verified through biochemical and serological testing. Of the serotypes chosen, Salmonella Kentucky, Dublin, and Enteritidis were the least resistant to CPC with a median minimum inhibitory concentration (MIC) of 14.22 μg/mL (range from 3.55 to 56.88 μg/mL); S. Typhimurium demonstrated a median MIC of 114.00 μg/mL (range from 28.44 to 114.00 μg/mL). Residual CPC potentially remaining attached to a carcass or in the weep after processing could potentially alter which Salmonella serotype is recovered from a carcass rinse due to different growth patterns during regulatory testing, with a potential for more virulent strains not to be recovered.


| INTRODUC TI ON
Salmonella enterica species are gram-negative rod-shaped bacteria commonly found in the gastrointestinal tract of poultry (Brenner et al., 2000) and are a primary cause of foodborne illness in the United States. It is estimated that 938 million cases of salmonellosis occur every year globally with non-typhoidal salmonellae as the cause (Antunes et al., 2016). Contaminated poultry products have been associated with approximately 25% of the outbreaks of foodborne salmonellosis (Akil & Ahmad, 2019). Contamination of poultry meat often occurs during processing from intestinal leakage (Berrang et al., 2001), contaminated processing equipment, water, and human contact. USDA-FSIS data show the prevalence of Salmonella in raw carcasses and parts has remained constant. Between October 2018 and September 2019, the presence of Salmonella was 8.77% and 3.62% for parts and carcasses, respectively (Food Safety and Inspection Service, 2020).
On January 1, 2006 the European Union (EU, 2003) and on January 1, 2017, the United States Food and Drug Administration (FDA) banned the use of antibiotics in chicken feed as growth promoters (AccessScience, s.v, 2017). This ban has led to changes in the way antimicrobial/biocidal compounds are used in the processing plants in the United States (Hudson & Sukumaran, 2020). Greater emphasis has been placed on postharvest strategies to decrease the microbial presence in carcasses. Increased use of antimicrobial chemicals is one of the most common interventions in poultry processing currently (Kim et al., 2017). Commonly used processing aids includes cetylpyridinium chloride (CPC), peroxyacetic acid (PAA), sodium hypochlorite, and acidified sodium chlorite.
Cetylpyridinium chloride is a water soluble, nonvolatile, quaternary ammonium compound (QAC) and has been found effective against various pathogenic bacteria without negatively affecting quality (Buncic & Sofos, 2012). Breen et al. (1995) reported that CPC (a quaternary ammonium compound) inhibited or reversed the attachment of Salmonella to chicken skin. Regulations require a water rinse and recapture of at least 99% of the antimicrobial for recycling ensuring that most of this antimicrobial is collected and not released into either the environment for small producers who may use collection ponds or into the local sewage/wastewater processing systems for larger producers who use municipal systems (Safe Foods Corporation, 2019).
It has been documented that QACs induce leakage of intracellular components which indicated membrane damage. CPC integrates into the lipid membrane of the bacterial cells which interferes with homeostasis and osmoregulation. The binding to anionic sites on the surface membranes of bacteria by CPC may cause slow or rapid cell death by damaging the cell walls resulting in leakage or solubilization of the cellular components depending on the concentration, low versus high (Morente et al., 2013). Paul et al. (2017) reported that both serotype and level of organic matter contamination may significantly influence bacterial survival. It is important to note that most studies conducted in vitro to determine efficacy of biocides typically use one serotype without evaluating the impact of the presence of the antibacterial activity of the biocide being tested (Rhouma et al., 2021). The objective of this study was to evaluate the effect of one biocide, CPC, on the recovery of Salmonella serotypes in a conventionally used medium supplemented with unknown factors (fats, proteins, etc.) from carcasses in vitro.

| Carcass rinsate
Ten carcasses were obtained from a local processing plant and transported back to the U.S. National Poultry Research Center, Russell Research Center pilot processing plant on ice in individual plastic bags (CryoVac®, SealedAir). Carcasses were transferred to clean individual CryoVac® bags and 400 mL of buffered peptone water (BPW, Accumedia, Neogen Culture Media) was added before being rinsed using a mechanical shaker (Simmons Engineering Company, Inc.) for 60 s. The rinsate was pooled into one container before being filtered through a prefilter (Whatman #4, Whatman Ltd) and filter sterilized using 0.2 μm PES Nalgene bottle top filter (Nalgene, Thermo Scientific). The sterilized carcass rinsate was stored at −80°C until use.

| Cetylpyridium chloride solution
A stock solution of CPC (2000 μg/mL, Safe Foods) was prepared in BPW with propylene glycol (3000 μg/mL, PG, Sigma Aldrich) to maintain CPC in solution and reduce absorption into treated poultry tissues (FDA, 2007). One milliliter of the filter-sterilized carcass rinsate was placed into all the wells of the 24-well tissue culture plate (Corning) and 1.0 mL of the CPC stock solution (2000 μg/mL of CPC/3000 μg/mL of PG) was placed into the first well (for an initial concentration of 1000 μg/mL CPC) and thoroughly mixed. Twelve wells were used per serotype with CPC concentrations from 909 to 0.44 μg/mL (Table 1)

| Salmonella isolates
Ten Salmonella serotypes (18 isolates, Table 2), six of which are commonly isolated from poultry and poultry products (Dr. Jonathan Frye, Bacterial Epidemiology and Antimicrobial Resistance Research Unit, U.S. NPRC), were selected for evaluation. All isolates were stored at −80°C in tryptic soy broth (TSB, Becton, Dickinson Co.) and 15% glycerol (Sigma-Aldrich Chemicals) until resuscitated for use.
Brilliant green agar with sulphapyridine (BGS, Neogen-Accumedia) was used for the cultivation and recovery of all Salmonella serotypes.
Inoculums of approximately 10 7 colony-forming units/mL (cfu/mL) were prepared by suspending each strain in sterile buffered peptone water (BPW, Neogen-Accumedia) and adjusting the absorbance to 0.20 ± 0.02 at 540 nm using a Spectronic 20D+ spectrophotometer (ThermoSpectronics, Thermo Scientific). This provides approximately 10 8 cfu/mL of each Salmonella strain which was followed by a 1:10 dilutions yielding approximately 10 7 cfu/mL of each strain. The strains were stored on ice until used to inoculate individual wells of 24-well tissue culture plates (Becton Dickinson Labware) which contained the diluted CPC/PG and filter-sterilized carcass rinsate.

| Antimicrobial assay
The tissue plates were inoculated with 100 μL of the individual serotypes into each of 12 wells and stored overnight at 4°C. After overnight storage, 10 μL from each well was streaked for isolation onto triplicate BGS agar plates and incubated 24 h at 37°C. After incubation, plates were observed for growth. The first dilution without growth was determined to be the minimum inhibitory concentration.
Colonies with typical morphology were stab inoculated into triple sugar iron (TSI, Becton, Dickinson Co.) and lysine iron (LIA, Becton, Dickinson Co.) agar slants for biochemical confirmation as Salmonella.
Serological confirmation was completed using Difco Salmonella Oantiserum (Becton, Dickinson Co.). Results are reported as minimum inhibitory concentration defined as the lowest concentration that will inhibit the growth of a microorganism after overnight incubation with confirmation on BGS plates without antibiotics and confirmed both biochemically and serologically. Three replicates were conducted, and the MIC concentrations are reported.  Table 3).

| RE SULTS AND D ISCUSS I ON
The additional strains of S. Kentucky and Infantis were selected because a previous study showed that these two serotypes were susceptible to the effect of CPC at very low concentrations (Cosby et al., 2018). In the present study, all 10 Salmonella serotypes exhibited growth at some level which contrasts with the data presented by Cosby et al. (2018) where S. Infantis (strain TA B L E 1 Dilution of chemicals (μg/mL) after addition of inoculum.  Addition of these two neutralizers at 1X their respective content in D/E buffer was required to allow for the recovery of Salmonella isolates after being exposed to 0.6% (6000 μg/mL) and 0.8% (

| CON CLUS IONS
In this study the effect of a novel substrate combined with a commonly used biocide on the recovery of Salmonella serotypes commonly associated with poultry and poultry products was investigated. The Salmonella serotypes selected for this study demonstrated a higher MIC when cultured in the rinsate than when grown in sterile BPW in a previous study. The mechanism of this higher resistance was not investigated in this study but will be investigated in future studies. This study demonstrated that additional factors found in the filter-sterilized rinsate might increase the ability of Salmonella serotypes to resist the action of commonly used biocides similar to the factors which would be present in a real-world carcass rinsate. These unknown factors might not be present when susceptibility testing is conducted using common media in a regulatory laboratory. Further research is necessary and planned to identify these unknown factors in order to improve the use of biocides as an intervention strategy for producers.

ACK N OWLED G M ENTS
The authors wish to acknowledge Ms. Lari Hiott and Ms. Susan Mize for the expert technical work and Mr. Dominic Arnold for his outstanding administrative support.

CO N FLI C T O F I NTE R E S T
None of the authors present a conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data will be available upon request from the Corresponding author.

E TH I C S S TATEM ENT
No human subjects or animals were utilized in this study.