Evaluation of Potassium-ClavulanateSupplemented Modified Charcoal-CefoperazoneDeoxycholate Agar for Enumeration of

Campylobacter in Chicken Carcass Rinse

Jung-Whan Chon, Hong-Seok Kim, Hyunsook Kim, Deog-Hwan Oh, and Kun-Ho Seo

Potassium-clavulanate-supplemented modified charcoal-cefoperazone-deoxycholate agar (C-mCCDA) that

was described in our previous study was compared with original mCCDA for the enumeration of Campylobacter in pure
culture and chicken carcass rinse. The quantitative detection of viable Campylobacter cells from a pure culture, plated on
C-mCCDA, is statistically similar (P > 0.05) to mCCDA. In total, 120 chickens were rinsed using 400 mL buffered
peptone water. The rinses were inoculated onto C-mCCDA and mCCDA followed by incubation at 42 C for 48 h.
There was no statistical difference between C-mCCDA (45 of 120 plates; mean count, 145.5 CFU/mL) and normal
mCCDA (46 of 120 plates; mean count, 160.8 CFU/mL) in the isolation rate and recovery of Campylobacter (P > 0.05)
from chicken carcass rinse. The Pearson correlation coefficient value for the number of Campylobacter cells recovered in
the 2 media was 0.942. However, the selectivity was much better on C-mCCDA than on mCCDA plates (P < 0.05).
Significantly fewer C-mCCDA plates (33 out of 120 plates; mean count, 1.9 CFU/mL) were contaminated with nonCampylobacter cells than the normal mCCDA plates (67 out of 120 plates; mean count, 27.1 CFU/mL). The C-mCCDA
may provide improved results for enumeration of Campylobacter in chicken meat alternative to mCCDA with its increased
selectivity the modified agar possess.

Keywords: Campylobacter, chicken carcass rinse, C-mCCDA, contaminants, enumeration

Introduction
Campylobacter spp. have been regarded as a major source of
gastrointestinal infection worldwide (Park and Sanders 1992;
Nachamkin and others 1998; Josefsen and others 2003). Campylobacter are a natural part of the intestinal flora in poultry (Josefsen
and others 2003; Belanger and Shryock 2007), and the number of
these bacteria in a carcass rinse can vary and, as a result, can affect
the number of these bacteria in a carcass rinse (Line 2001a; Line
and Berrang 2005; Oyarzabal and others 2005). Enumeration of
Campylobacter on chicken carcasses by using sensitive and quantitative detection methods has been a major goal for many food
quality control authorities (Josefsen and others 2003).
Although there are various quantitative methods to detect
Campylobacter that do not involve culturing of the bacteria, direct
plating onto selective agars is still regarded as an effective technique for isolating and enumerating Campylobacter from a carcass
rinse, as shown in previous studies (Line 2001; Line and Berrang
2005; Oyarzabal and others 2005). However, the differentiation of
suspected Campylobacter colonies from other contaminating species
by using selective agars are often confounded by an overgrowth
of non-Campylobacter colonies (Line 2001; Line and others 2001;
Line and Berrang 2005; Line 2006; Jasson and others 2009; Chon
and others 2012a).
MS 20131378 Submitted 9/27/2013, Accepted 12/23/2013. Authors Chon, HS Kim, H Kim, and Seo are with KU Center for Food Safety, College of Veterinary
Medicine, Konkuk Univ., Seoul, The Republic of Korea. Author Oh is with Dept.
of Food Science and Biotechnology, Inst. of Bioscience and Biotechnology, Kangwon
Natl. Univ., Chuncheon, Kangwon 200-701, Republic of Korea. Direct inquiries to
author Seo (E-mail: bracstu3@konkuk.ac.kr).
Both

authors contributed equally to this work.

R

C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12388
Further reproduction without permission is prohibited

Most selective agars commonly used in food quality control are supplemented by a high concentration of cefoperazone, a 3rd-generation cephalosporin (Corry and others 1995).
However, resistance to cefoperazone has recently become more
widespread in food-related bacteria, causing a failure of isolation of
Campylobacter spp. in raw poultry meat (Jasson and others 2009;
Moran and others 2011; Chon and others 2012a). In particular, extended spectrum beta-lactamase (ESBL)-producing Escherichia coli may grow on selective agar because of its resistance
to cephalosporins. The ESBL-producing bacteria have frequently
been isolated in raw chicken in many countries, and have become
a common factor affecting isolation and precise enumeration of
Campylobacter in chicken meat (Warren and others 2008; Costa and
others 2010; Leverstein-van Hall and others 2011; Moran and others 2011). Therefore, eliminating the growth of ESBL-producing
bacteria on selective media increases both the sensitivity and selectivity of Campylobacter-selective agar.
Modified charcoal-cefoperazone-deoxycholate agar (mCCDA)
has frequently been used by many microbiologists for detection
and enumeration of Campylobacter in clinical and food samples
(Corry and others 1995; Engberg and others 2000; Oyarzabal
and others 2005; Moran and others 2011). Rosenquist and others
(2007) reported that the direct plating on mCCDA is an acceptable
method for quantitative detection of Campylobacter in chicken meat
(Habib and others 2011). mCCDA is formally used in Intl. Organization for Standardization (ISO) for enumeration of Campylobacter
in foods (ISO 2006; Ahmed and others 2012). However, mCCDA
is prone to contamination by ESBL-producing bacteria, thereby
rendering differentiation of suspected Campylobacter colonies difficult (Moran and others 2011; Chon and others 2012a, 2012b). In
a previous study, we described an improved mCCDA through the

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Abstract:

Enumeration of Campylobacter with C-mCCDA . . .

addition of 0.5 mg/L of potassium clavulanate, an ESBL inhibitor,
and qualitatively evaluated the detection of Campylobacter from
chicken carcass rinse on the modified agar (potassium-clavulanatesupplemented mCCDA; C-mCCDA) (Chon and others 2013).
The modification of mCCDA resulted in greater (P < 0.05) isolation rate and selectivity than did the original mCCDA (Chon and
others 2013). However, enumeration of Campylobacter by using
C-mCCDA was not evaluated in that study.
In this study, we quantitatively evaluated the detection of
Campylobacter in C-mCCDA and compared it with detection in
the original mCCDA. The Campylobacter used in this study were
obtained from pure culture and from chicken carcass rinses. The
most prominent competing microflora on both mCCDA and CmCCDA from chicken carcasses were also identified.

Materials and Methods

Table 1The mean number of cells from the 6 Campylobacter

strains, each of the 3 C. jejuni and the 3 C. coli strains, recovered on the 2 selective agars.
Nr. of recovered cells
(CFU/0.1 mL) on plate
Species
C. jejuni
C. coli
Mean SEMa

Inoculum
(CFU/0.1 mL)

C-mCCDA

mCCDA

113.0 25.4
115.3 44.1
114.2 22.8

79.0 13.0
59.7 19.6
69.3 11.4A

68.3 19.7
61.0 32.2
64.7 12.2A

Statistically significant differences among values in rows are indicated by differing

uppercase letters (P < 0.05).

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Biochemical confirmation of contaminants on plates

To identify the competing flora frequently appeared on the 2 selective agars, we biochemically confirmed contaminants from each
of mCCDA and C-mCCDA in the 6th trial. In the 6th trial, with
20 chicken carcasses, we subcultured a single contaminant that
appeared on each selective agar. In total, 14 subcultured colonies
(4 contaminant colonies from 4 C-mCCDA plates and 10 contaminant colonies from 10 mCCDA plates) were biochemically
identified using the Vitek 2 GN kit (bioMerieux, Marcy lEtoile,
France). The ESBL enzyme production was also tested using the
Vitek 2 AST n224 kit (bioMerieux) according to manufacturers
recommendations.

Bacterial strains
In total, 6 strains of Campylobacter (3 Campylobacter jejuni strains:
NCTC 11168, CJ_Yim2 [human isolate], and GD_4 [chicken
isolate]; and 3 C. coli strains: SD_4 [chicken isolate], CCHS12-2
[chicken isolate], and DD_2 [chicken isolate]) were used in the
current study. All Campylobacter strains were from our own collection. Campylobacter strains kept frozen at 70 C were inoculated
on blood agar (Oxoid, Hampshire, U.K.), followed by incubation
microaerobically (5% O2 , 10% CO2 , and 85% N2 ) at 37 C for
48 h for 2 passages.
Data analysis
Statistical analyses were conducted using SPSS, version 19.00.
Differences in the number of recovered Campylobacter cells and
Preparation of mCCDA and C-mCCDA
The mCCDA (Oxoid) was prepared according to the manufac- non-Campylobacter contaminants from pure culture and chicken
turers instructions. With reference to our previous study (Chon carcass rinse on the 2 selective agars were compared using a tand others 2013), C-mCCDA was made by adding 0.5 mg of test. The Pearson correlation coefficient was also used to compare
potassium clavulanate (Sigma, St. Louis, Mo., U.S.A.) to 1 L of the accuracy of enumeration by using the 2 selective agars. We
cooled mCCDA. All prepared plates were stored at a low temper- compared the number of plates positive for Campylobacter and
non-Campylobacter contaminants. The plates were compared using
ature (4 C) and used within 5 d.
Fishers exact test in pairs.
Campylobacter from pure culture recovered using mCCDA
and C-mCCDA
We compared the number of colonies recovered from each of the
2 agars, starting from pure cultures. A single colony each of the 3 C.
jejuni and the 3 C. coli strains described above were inoculated into
20 mL of Bolton broth (Bolton broth) and incubated at 37 C for
48 h under microaerobic condition. The incubated Bolton broth
was 10-fold serially diluted in saline water, and 0.1 mL of each of
these dilutions of pure culture with a cell count ranging from 30
to 300 were inoculated onto mCCDA and C-mCCDA. All plates
in duplicates were incubated at 37 C for 48 h microaerobically
and colonies are enumerated.
Isolation of Campylobacter from chicken carcass rinse
In total, 120 chickens were purchased between May 2012 and
May 2013 from 5 different retail stores located in Seoul, South
Korea. Chicken carcasses were rinsed with 400 mL of buffered
peptone water (Difco, Sparks, Md., U.S.A.) by gentle shaking up
to 1 min. After shaking, 0.1 mL of the wash was 10-fold serially
diluted in saline water, and each dilution (0.1 mL) was inoculated
onto the 2 selective agars in duplicates. Plates were incubated at
42 C for 48 h under microaerobic condition. Suspected Campylobacter colonies (up to 5 colonies from each plate) were subcultured onto blood agar and finally confirmed using colony PCR
with reference to Denis and others (1999).
M924 Journal of Food Science r Vol. 79, Nr. 5, 2014

Results and Discussion

Recoverability of Campylobacter from pure culture using
mCCDA and C- mCCDA
Numbers of recovered cells of Campylobacter on the 2 selective
agars from pure culture are compared in Table 1. There was no
statistically significant difference (P > 0.05, Table 1) in the number
of Campylobacter cells between the 2 selective agars. This result
indicates that the quantitative detection of viable Campylobacter
cells from a pure culture, plated on C-mCCDA, is statistically
similar (P > 0.05) to mCCDA.
Comparison of performance of the 2 selective agars
Data on Campylobacter and non-Campylobacter cells recovered
from the 2 selective media from chicken carcass rinses are presented
in Table 2 and 3. The number of C-mCCDA and mCCDA plates
positive for Campylobacter was similar, with no statistical difference
(C-mCCDA, 45 of 120 [37.5%]; mCCDA, 46 of 120 [38.3%];
Table 2). Populations of Campylobacter recovered from a milliliter
of the chicken carcass washes are shown in Figure 1. As determined
by direct plating, no statistical difference was found between the
2 agars (P > 0.05), although C-mCCDA exhibited slightly lower
mean counts (145.5 CFU/mL) than mCCDA (160.8 CFU/mL,
Table 3). The Pearson correlation coefficient value for the number
of recovered Campylobacter cells between the 2 media was 0.942,

Enumeration of Campylobacter with C-mCCDA . . .

Nr. of positives/
total number of samples (%)
Strains
Campylobactera
Non-Campylobactera

C-mCCDA

mCCDA

45/120A

46/120A (38.3)
67/120B (55.8)

(37.5)
33/120A (27.5)

Statistically significant differences among values in rows are indicated by differing

uppercase letters (P < 0.05).

Table 3Comparison of the number of Campylobacter and nonCampylobacter cells recovered on C-mCCDA and mCCDA from
120 chicken carcass rinses.
Strains
Campylobacter
Non-Campylobacter

Value (CFU/mL)
Mean
Range
Mean SEMa
Range
SEMa

C-mCCDA
145.5
0 to 2125
1.9 0.3A
0 to 20

36.0A

mCCDA
160.8 38.9A
0 to 2750
27.1 8.1B
0 to 670

Statistically significant differences among values in rows are indicated by differing

uppercase letters (P < 0.05).

indicating that the quantitative detection of these bacteria was

similar in the 2 selective agar media (Figure 1).
However, the selectivity was much better (P < 0.05) on
C-mCCDA than on mCCDA as judged by the fewer number of competing microflora. Significantly fewer (P < 0.05)
C-mCCDA (33 of 120) plates were contaminated with nonCampylobacter strains than normal mCCDA plates were (67 of
120). A mean of 1.9 CFU/mL non-Campylobacter was found on
C-mCCDA, compared to a mean of 27.1 CFU/mL on mCCDA
(P < 0.05).
Although mCCDA agar is the most popular selective agar media for the isolation of Campylobacter in poultry samples, the dense

for isolation of the pathogen (Jasson and others 2009; Moran

and others 2011; Chon and others 2012a; Hayashi and others
2013). Many researchers reported that ESBL-producing E. coli
may grow profusely on agars. Such contamination can mask suspicious Campylobacter and lead to false positives or negatives, rendering differentiation and enumeration difficult (Jasson and others
2009; Moran and others 2011; Ahmed and others 2012; Chon
and others 2012a; Hayashi and others 2013).
In the 6th trial (n = 20), we identified 14 contaminants
from the 2 selective agars. All tested colonies isolated from CmCCDA (4 colonies) were identified as Acinetobacter spp. (4 of 4,
100%). Of the 10 contaminant colonies subcultured from mCCDA, only 2 were Acinetobacter spp. (2 of 10, 20%), and the
remainder were E. coli (8 of 10, 80%). All E. coli strains were
positive for the ESBL enzyme. It appears that ESBL-producing
E. coli were effectively eliminated on C-mCCDA owing to the
presence of potassium clavulanate.
Previous studies have demonstrated the efficacy of addition of
an ESBL inhibitor for selective isolation of Campylobacter from
poultry meat. Moran and others (2011) added potassium clavulanate to the Bolton broth, and they reported improved Campylobacter isolation rates. We have also reported that potassiumclavulanate-containing mCCDA is more suitable than normal
mCCDA for isolation of Campylobacter from chicken carcass
rinse (Chon and others 2013). However, to our knowledge,
no study has evaluated the effect of potassium clavulanate on
the quantitative detection of Campylobacter from chicken carcass
rinse without an enrichment step. In this study, we evaluated
the quantitative detection of Campylobacter from chicken carcass
rinse in C-mCCDA and normal mCCDA, which has frequently
been used for the enumeration of Campylobacter (Line and others 2001; Oyarzabal and others 2005; Rosenquist and others
2007).

Figure 1Campylobacter recovered from

carcass rinse samples using the 2 selective
agars C-mCCDA and mCCDA represented
as a correlation coefficient.

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Table 2Comparison of the number of plates of C-mCCDA and growth of non-Campylobacter on the cefoperazone-containing
mCCDA positive for Campylobacter and non-Campylobacter strains agars has been one of the most common confounding factors
from 120 chicken carcass rinses.

Enumeration of Campylobacter with C-mCCDA . . .

Conclusion
According to our findings, although C-mCCDA provided
greater (P < 0.05) selectivity than normal mCCDA, quantitative detection ability of C-mCCDA to recover Campylobacter cells
in pure culture and chicken carcass rinse is statistically similar (P >
0.05) to that of mCCDA. Our results indicate that the increased
selectivity of C-mCCDA may be a useful tool in the enumeration
of Campylobacter cells in carcass rinse samples.

Acknowledgments
This research was supported by Mid-career Researcher Program
(2012R1A2A2A-01015344) through the Natl. Research Foundation of Korea (NRF) funded by the Ministry of Education,
Science, and Technology, and by the Export Promotion Technology Development Program of iPET (nr. 313010-3) funded by
the Ministry for Food, Agriculture, Forestry, and Fisheries. JungWhan Chon and Hong-Seok Kim were also partially supported
by the Brain Korean 21 (BK21) Project from the Ministry of
Education and Human Resources Development.

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