is the main cause of bacterial gastroenteritis in the world and researchers
have shown it to be present in high levels in retail poultry. , Of Campylobacter illnesses, 50 - 70% were
caused by consumption of poultry or poultry products. , Symptoms of Campylobacter include fever, abdominal
pain and diarrhea within 2 - 5 days of ingesting contaminated product. ,
Stricter performance standards have been implemented by the USDA, aimed at
reducing incidence of Campylobacter
in processing facilities.  FSIS estimated 46% of poultry facilities would
not be able to meet new requirements; therefore, processors must give serious attention
to finding efficient antimicrobial treatments to be used throughout the plant.
It is mandatory Campylobacter
prevalence remain below 7.7% on poultry parts (4 of 52 samples), allowing only
a small margin of error. 
A variety of antimicrobials have
been evaluated for use on poultry at various points throughout processing
procedures. It is essential chemicals be cost effective, reduce pathogen
prevalence and not cause organoleptic damage to poultry carcasses or parts. 
Antimicrobials vary in levels of efficacy, treatment concentration, contact
time and application method. Chemical efficacy is affected by microbial load,
composition of flora, organic material or possibly changed from residual
effects of prior treatments.  Stopforth et al.  determined a single
intervention could not significantly reduce pathogen presence on finished
carcasses. Instead, a multi-hurdle approach is required at various intervention
points throughout processing for increased pathogen reduction on poultry products.
, There was no previous research found evaluating consecutive,
sequential treatments and how they affect pathogens. The purpose of this study
is to evaluate the effectiveness of a low acid antimicrobial, CMS PoultrypHreshä, and whether dipping carcasses in consecutive or sequential
treatments along with peracetic acid (PAA) leads to further reductions in the
prevalence of Campylobacter on
Bacterial Marker Strain
Cox et al.  developed a marker
strain of Campylobacter resistant to
the antibiotic gentamicin, Campylobacter
coli (CCGR), which was used in this study. Campy Cefex agar
(Sigma, St. Louis, MO) plates were prepared using 200 ppm gentamicin (CCGen) to
eliminate the growth of wild type Campylobacter
that may be naturally present on poultry thighs. Initial cultures were streaked
from storage in an -80°C
freezer, maintained in Bolton’s broth with 15% glycerol and no supplements.
Cultures were streaked onto CCGen agar and incubated in sealed bags under
microaerobic (5% O2, 10% CO2, 85% N2)
conditions at 42°C
for 48 h. A sterile swab was used to remove colonies and mix them into a 9 mL
tube of Bolton’s broth, placed in sealed bags and incubated microaerobically
for 24 h at 42°C. Tubes were evaluated for colorimeter
analysis by a Spectronic 200E (Thermo Fisher Scientific, Madison, WI) and
approximately a 108 cfu/mL CCGR inoculum was prepared.
Inocula were confirmed using serial dilutions, plated on CCGen and incubated
for 48 h at 42°C.
Skin-on poultry thighs were
purchased from a local grocery (n=18) and divided into the six treatment
groups. A 6.5 cm2 section of skin was inoculated on each thigh by
spreading 0.1 mL of 108 CCGR marker strain. Each thigh
was inoculated individually and given a 5 min attachment period before
treatment. Thighs were individually dipped into Ziploc bags containing either 1
liter of water, PAA (600 ppm), or PoultrypHresh (pH 1.5) for 6 s. Thighs were
removed and placed on tin foil squares for 60 s. Each thigh was then dipped
into the second dip treatment for 6 s, drained 5 s, placed into individual
sealable bags with 150 mL buffered peptone water (BPW), and hand shaken and
massaged for 60 s. Thighs were removed, and rinses placed on ice for
approximately 15 min before diluting. Three thighs served as controls in each
replication and were inoculated as described, remained untreated and placed
directly into rinse bags with BPW after the attachment period. The entire
experiment was replicated 5 times.
Plating and Incubation
Rinsates were serially diluted and a
plate spreader used to disperse onto CCGen agar plates in duplicate. Plates
were incubated at 42°C
for 48 h. Characteristic colonies were counted, and cfu/mL log transformed.
The study was constructed of five
replicates of skin-on thighs using 18 individual thighs (N=18; n=3). Each
replicate consisted of 3 of each untreated, consecutive water dips, consecutive
PoultrypHresh™ (pH 1.5) dips, consecutive PAA (600 ppm) dips, PoultrypHresh™
followed by PAA dip, and PAA followed by PoultrypHresh™ dip. Duplicate counts
were averaged, and numbers were transformed by log10. Data was
analyzed using Statistica software (Statistica, 2013). A General Linear Model
was conducted to determine whether sequential dips were statistically different
(P < 0.05). Means were separated
with a Tukey Multiple Comparison test; statistical significance was assigned at
P = 0.05.
water dips were used to determine rinsing effect of water alone. A 0.8 log10
cfu/mL Campylobacter reduction
was observed, although contamination remained high at 4.9 log10
cfu/mL (Table 1). Treating with consecutive PoultrypHreshä dips significantly (P
= 0.05) reduced Campylobacter from
5.64 log10 cfu/mL untreated and 4.87 log10 cfu/mL water
dipped to 3.90 log10 cfu/mL (Table 1). This equates to a reduction
of 98.2% from untreated and 89.3% from water dipped thighs (Table 1). Dipping
with consecutive PAA dips showed slightly higher reductions; reducing Campylobacter 99.3% (2.1 log10
cfu/mL) from untreated and 95.7% (1.4 log10 cfu/mL) from water
dipped samples (Table 1).
Dipping in PoultrypHreshä followed by PAA demonstrated findings similar to
consecutive PAA dips. Interestingly, thighs dipped in PAA followed by
PoultrypHreshä, showed reductions significantly (P = 0.05) greater than any other dipping
combination. Campylobacter was
reduced 99.9% (> 3 log10 cfu/mL reduction) from untreated thighs
and 99.6% (2.4 log10 cfu/mL reduction) from water treated (Table 1).
This pattern was observed in all 5 replications of the study; therefore, the
order of chemicals used in dipping sequences was significantly (P = 0.05) correlated to Campylobacter reductions.
can be compared to a study by Landrum et al. (2018) evaluating PoultrypHresh™
on broiler thighs, where Campylobacter reductions
were higher compared to untreated from this study, 2.2 log10 cfu/mL
and1.7 log10 cfu/mL, respectively. Comparing the water dipped thighs
to untreated, the research by Landrum et al. (2018) demonstrated a 1.4 log10
cfu/mL reduction, whereas this study a 1.0 log10 cfu/mL. Differences
between the two studies were the dip time, (25 s compared to two 6 s dips) and
air agitation, not used in this study. Shorter dip times of 6 s were chosen for
this research as a more practical time of exposure in a modern processing
facility. The higher dip times used by Landrum et al. (2018) exhibited better
results, seemingly due to the longer exposure time, although the effects of air
agitation could have also assisted in higher reductions. The improved microbial
reductions using air agitation have been shown in previous research. 
Therefore, air agitation is a concept that may require more research to heighten
reductions of microbial contamination.
is a low pH organic peroxide mixture of acetic acid and hydrogen peroxide
currently being used throughout the United States in multiple processing
facilities. ,, The mode of action for PAA is a disruption to the
cell membrane permeability, altering protein synthesis, leading to bacterial
death.  PAA reduced the presence of Campylobacter
slightly more than consecutive PoultrypHreshä dips, although differences were not significant. Findings
by Bauermeister et al.  showed a 1.5 log10 cfu/mL Campylobacter reduction using an
extremely low concentration of only 200 ppm PAA. As PAA is approved for use
throughout processing at concentrations up to 2000 ppm, reductions could be
even greater with increased acid levels.  Bauermeister  used 200 ppm
PAA, only demonstrating a 1.5 log10 cfu/mL reduction; therefore,
other factors could be associated to its efficacy. King et al.  determined
that the effects of PAA may vary and greatly depend on bacteria level and how
they are attached to the surface. Nagel et al.  evaluated higher levels of
PAA at 400 and 1000 ppm and reduced Campylobacter
levels by more than 2.0 log10 cfu/mL.
have determined a multi-hurdle approach is necessary for adequate pathogen
reduction, which uses multiple intervention points throughout processing
procedures such as the inside-outside bird washer, brush washer, cabinet
washer, or dip tank before and/or after chilling.  Using this approach, the
facility does not rely only on a single step intervention, but instead
incorporates many applications for reducing foodborne pathogen prevalence prior
to entering secondary processing ,, No research was found, however,
demonstrating the usefulness of consecutive, sequential chemical hurdles for
pathogen reduction. This study demonstrates the possibility that consecutive
application of chemicals in a specific sequential order may reduce Campylobacter prevalence.
Table 1. Average Log10
cfu/mL of Campylobacter coli
recovered by replicate from sequentially dip treated thighs with no treatment,
water-water, PpH-PpH, PAA-PAA, PpH-PAA, or PAA-PpH (mean ± standard error).
Average Log10 cfu/mL
Reduction compared to Untreated (%)
Reduction compared to Water (%)
1 PpH = CMS PoultrypHresh™ 2 PAA = peracetic acid
from this study demonstrated PoultrypHreshä and PAA could reduce the prevalence of Campylobacter on poultry parts when used separately. However, when
evaluated sequentially, PAA followed by PoultrypHreshä can significantly reduce pathogen presence. Such findings
may be extremely beneficial and lead to better intervention strategies in
future antimicrobial applications.
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