**1. Introduction**

The Foodborne Diseases Active Surveillance Network (FoodNet) of the Centers for Disease Control and Prevention's (CDC) Emerging Infections Program reported that in 2019, *Campylobacter* was the leading bacterial cause of foodborne illness with an incidence rate of 19.5 cases per 100,000 population [1]. Specifically, out of 25,866 total cases of foodborne illness that were laboratory-diagnosed in that year, 9731 were due to infection with *Campylobacter* [1]. Individuals with *Campylobacter* infections, however, do not always seek medical treatment and even if they do, cases may remain undiagnosed [2]. Therefore, when underreporting and underdiagnosis are factored in, estimates indicate that *Campylobacter* spp. are actually responsible for 1.5 million diarrheal illnesses each year in the United States [2,3]. The most common species of *Campylobacter* associated with human campylobacteriosis cases is *C. jejuni*, and is responsible for at least 80% of *Campylobacter* enteric infections [4,5].

*Campylobacter* infections are primarily associated with consumption of unintentionally undercooked contaminated poultry products [6,7]. Moreover, *Campylobacter* in poultry is the number one pathogen-food combination in terms of annual illness burden, with a total of 608,231 infections and an estimated cost of more than \$1.2 billion [8]. In an effort to reduce the incidence of foodborne illness cases from poultry products, slaughter facilities are required by the U.S. Department of Agriculture's (USDA) Food Safety and

**Citation:** Gonzalez, S.V.; Geornaras, I.; Nair, M.N.; Belk, K.E. Evaluation of Immersion and Spray Applications of Antimicrobial Treatments for Reduction of *Campylobacter jejuni* on Chicken Wings. *Foods* **2021**, *10*, 903. https://doi.org/10.3390/foods10040903

Academic Editor: Huerta-Leidenz Nelson and Markus F. Miller

Received: 18 March 2021 Accepted: 16 April 2021 Published: 20 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Inspection Service (FSIS) to identify points during slaughter and processing where physical and/or chemical interventions can be applied to reduce pathogen contamination levels [9]. In the United States, FSIS Directive 7120.1 [10] provides poultry processors with a list of antimicrobials that are approved for use as decontamination treatments of poultry products. Peroxyacetic acid (PAA), which is currently the most widely used antimicrobial intervention in U.S. poultry processing facilities, is approved for use up to a maximum concentration of 2000 ppm [10,11]. Also approved are various organic and inorganic acids, cetylpyridinium chloride, chlorine, acidified sodium chlorite, trisodium phosphate, and a blend of sulfuric acid and sodium sulfate (SSS; also referred to as AFTEC 3000 or Amplon in the literature) [10,11]. SSS can be used as a spray, immersion, or wash treatment of poultry products at concentrations that would achieve a targeted pH range of 1.0 to 2.2 [10].

Performance standards for *Salmonella* and *Campylobacter*, established by FSIS, are used to assess the effectiveness of decontamination interventions used by a facility, in limiting or reducing pathogen contamination [12]. Since more than 85% of poultry meat in the United States is sold as parts, FSIS includes in its testing program sampling sites for both pathogens in the cut-up room to test poultry parts [13,14]. The current performance standards for the maximum acceptable *Campylobacter*-positives for chicken are 15.7% of broiler carcasses, 9.6% of comminuted products, and 7.7% of parts [13,15]. Thus, the poultry industry is reevaluating current antimicrobial interventions used for pathogen control and is looking for novel decontamination treatments to apply to meet the strict regulations for poultry [14,16].

There are numerous published studies on the antimicrobial effects of various chemical treatments against *Salmonella* populations on whole chicken carcasses and parts [11,14,17–19]. In comparison, however, fewer research studies have reported on the effect of such treatments against *Campylobacter*, and in particular, on chicken parts. Additionally, regardless of poultry product type and pathogen, studies investigating the decontamination efficacy of chemical treatments that combine two or more modes of action are also limited. Therefore, the objectives of this study were to (i) evaluate the antimicrobial effects of SSS, formic acid, PAA, and PAA that was pH-adjusted with SSS or formic acid (hereafter referred to as "acidified PAA"), when applied to chicken wings inoculated with *C. jejuni*, and (ii) determine the antimicrobial efficacy of the treatments as a result of applying the test solutions by immersion or spraying. Additionally, the antimicrobial effects against inoculated populations were evaluated immediately after treatment application (0 h) and after 24 h of storage at 4 ◦C.

### **2. Materials and Methods**

### *2.1. Bacterial Strains and Inoculum Preparation*

The inoculum consisted of a mixture of six *C. jejuni* strains of poultry origin (Table 1). Working cultures of the strains were maintained at 4 ◦C on plates of Campy Cefex Agar, Modified (mCCA; Hardy Diagnostics, Santa Maria, CA, USA) that were held within anaerobic containers (AnaeroPack Rectangular Jar; Mitsubishi Gas Chemical America, New York, NY, USA) with a microaerophilic environment generating gas pack (mixture of approximately 6 to 12% O2 and 5 to 8% CO2; AnaeroPack-MicroAero sachet, Mitsubishi Gas Chemical America).


**Table 1.** *Campylobacter jejuni* strains used in the study.

a U.S. Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science. b U.S. Food and Drug Administration, Center for Veterinary Medicine.

The *C. jejuni* strains were individually cultured and subcultured in 10 mL of Bolton broth (Hardy Diagnostics) incubated at 42 ◦C for 48 h under microaerophilic conditions (Oxoid CampyGen sachet, Thermo Scientific, Basingstoke, UK). Cultures of the six strains were then combined and centrifuged (6000× *g*, 15 min, 25 ◦C; Sorvall Legend X1R centrifuge, Thermo Scientific, Waltham, MA, USA). Resulting cell pellets were washed twice with 10 mL of phosphate-buffered saline (PBS; pH 7.4; Sigma-Aldrich, St. Louis, MO, USA), and the final washed cell pellet comprising all six strains was resuspended in 60 mL of PBS. This cell suspension (ca. 7 log colony-forming units [CFU]/mL concentration) was then diluted 10-fold in PBS, and the diluted inoculum (ca. 6 log CFU/mL concentration) was used to inoculate the chicken wings. The concentration of the *C. jejuni* inoculum (undiluted and diluted) was determined by plating serial dilutions onto mCCA.

### *2.2. Inoculation of Chicken Wings*

Fresh (i.e., not frozen) skin-on whole chicken wings were purchased from a wholesale food distributor. Wings were stored at 2 ◦C and were used for the study within six days of receipt. Two trials (repetitions) of the study were conducted on two separate days. On the first day of each trial, wings were randomly assigned to a control treatment or one of six treatments to be applied by immersion or spraying. For each antimicrobial treatment and application method, six samples were placed on trays lined with ethanol-sterilized aluminum foil and were inoculated under a biological safety cabinet. A 0.1 mL (100 μL) aliquot of the diluted *C. jejuni* inoculum was deposited, with a micropipette, on one side of each wing and then spread over the entire surface with a sterile disposable spreader. After a 10 min bacterial cell attachment period, samples were turned over, with sterile forceps, and were inoculated on the second side using the same procedure. The second inoculated side was also left undisturbed for 10 min to allow for inoculum attachment. The target inoculation level was 3 to 4 log CFU/mL of wing rinsate.

### *2.3. Antimicrobial Treatment of Chicken Wings*

Inoculated wings were left untreated, to serve as controls, or they were treated by immersion or a spray application with water, SSS (pH 1.2; Amplon, Zoetis, Florham Park, NJ, USA), formic acid (1.5%; BASF Corporation, Florham Park, NJ, USA), PAA (550 ppm; Actrol Max, Kroff, Pittsburgh, PA, USA), PAA (550 ppm) acidified with SSS (pH 1.2; SSS-aPAA), or PAA (550 ppm) acidified with formic acid (1.5%; FA-aPAA). The water treatment was included to determine the rinsing effect of the immersion and spray treatments. Antimicrobial treatment solutions were prepared according to the manufacturers' instructions, and the pH of solutions was measured (Orion Star A200 Series pH meter and Orion RossUltra pH electrode, Thermo Scientific, Schaumburg, IL, USA). Average pH values of the SSS, formic acid, and PAA solutions were 1.2, 2.9, and 3.2, respectively. For the SSS-aPAA and FA-aPAA solutions, average pH values were 1.2 and 2.8, respectively. The PAA concentration was verified using a hydrogen peroxide and peracetic acid test kit (LaMotte Company, Chestertown, MD, USA).

For immersion application of the test solutions, inoculated wings were individually immersed for 5 s in 500 mL of the solution in a Whirl-Pak bag (1627 mL; Nasco, Fort

Atkinson, WI, USA). A different Whirl-Pak bag and fresh, unused solution was used to immersion-treat each sample. Spray application of the water and chemical treatments was performed using a custom-built spray cabinet (Birko/Chad Equipment, Olathe, KS, USA) fitted with two 0.38 L/min FloodJet spray nozzles (Spraying Systems Co., Glendale Heights, IL, USA) positioned above the product belt. The inoculated wings were placed on a cutting board on top of the ladder-style conveyor belt of the cabinet and were sprayed with the test solution at a pressure of 69 to 83 kPa and a product contact time of 4 s.

Immersion- and spray-treated wings were placed on sterile wire racks for 5 min to allow excess solution to drip off samples before microbiological analysis or refrigerated storage. For each trial, three of the six samples per treatment were analyzed for *C. jejuni* populations following treatment application (0 h analysis), and the three remaining samples were placed in individual 710 mL Whirl-Pak bags (Nasco) and analyzed after a 24 ± 1 h storage period at 4 ◦C.

### *2.4. Microbiological Analysis*

At each sampling time (0 h and 24 h), untreated (control) and treated samples were analyzed for *C. jejuni* populations. For microbial analysis of 0 h samples, wings were placed in a Whirl-Pak bag (710 mL) containing 150 mL of neutralizing buffered peptone water (nBPW; Acumedia-Neogen, Lansing, MI, USA) [20]. For the 24 h samples, which were already in Whirl-Pak bags, 150 mL of nBPW was aseptically poured into each bag. Sample bags containing individual wings were vertically shaken by hand with a strong downward force, 60 times, to recover cells from the wing surface. Rinsates were serially diluted (1:10) in buffered peptone water (Difco, Becton Dickinson and Company, Sparks, MD, USA) and appropriate dilutions were surface-plated, in duplicate, onto pre-warmed (42 ◦C) mCCA plates. Plates were placed into anaerobic containers (AnaeroPack Rectangular Jar) with an appropriate number of microaerophilic environment generating gas packs (AnaeroPack-MicroAero), per manufacturer instructions, and were incubated at 42 ◦C for 48 ± 1 h. Three uninoculated and untreated chicken wings were also analyzed on each of the inoculation and treatment application days, for natural microflora counts (on Tryptic Soy Agar [Acumedia-Neogen]; 25 ◦C for 72 h) and for any naturally-present *Campylobacter* populations (on mCCA) on the chicken wings used in the study. The detection limit of the microbiological analysis was 1 CFU/mL.

### *2.5. Statistical Analysis*

The study was designed as a 7 (treatments) × 2 (sampling times) factorial for each solution application method (immersion, spraying), blocked by trial day. It was repeated on two separate days, and three samples were analyzed per treatment and sampling time (0 h and 24 h) in each trial (i.e., a total of six samples per treatment and sampling time). For each solution application method, recovered *C. jejuni* populations were statistically analyzed across all treatments within each sampling time (0 h, 24 h), and across the two sampling times for each antimicrobial treatment. Bacterial populations were expressed as least squares means for log CFU/mL of wing rinsate under the assumption of a log-normal distribution of plate counts. Data were analyzed using the emmeans package [21] in R (version 3.5.1). Means were separated with Tukey adjustment using a significance level of α = 0.05.
