A simplified and cost-effective enrichment protocol for the isolation of Campylobacter spp. from retail broiler meat without microaerobic incubation
A simplified and cost-effective enrichment protocol for the isolation of Campylobacter spp. from retail broiler meat without microaerobic incubation
Ping Zhou 0
Syeda K Hussain 2
Mark R Liles 1
Covadonga R Arias 4
Steffen Backert 3
Jessica Kieninger 0
Omar A Oyarzabal 0
0 Department of Biological Sciences , 1627 Hall Street , Alabama State University , Montgomery, AL , USA
1 Department of Biological Sciences, 101 Rouse Life Science Bldg Auburn University , AL , USA
2 Department of Microbiology and Immunology, University of Arkansas for Medical Sciences , Little Rock, AK , USA
3 University College Dublin, UCD School of Biomolecular and Biomedical Sciences, Science Center West , Belfield Campus, Dublin 4 , Ireland
4 Department of Fisheries and Allied Aquacultures, 101 Swindle Hall, Auburn University , AL , USA
Background: To simplify the methodology for the isolation of Campylobacter spp. from retail broiler meat, we evaluated 108 samples (breasts and thighs) using an unpaired sample design. The enrichment broths were incubated under aerobic conditions (subsamples A) and for comparison under microaerobic conditions (subsamples M) as recommended by current reference protocols. Sensors were used to measure the dissolved oxygen (DO) in the broth and the percentage of oxygen (O2) in the head space of the bags used for enrichment. Campylobacter isolates were identified with multiplex PCR assays and typed using pulsed-field gel electrophoresis (PFGE). Ribosomal intergenic spacer analyses (RISA) and denaturing gradient gel electrophoresis (DGGE) were used to study the bacterial communities of subsamples M and A after 48 h enrichment. Results: The number of Campylobacter positive subsamples were similar for A and M when all samples were combined (P = 0.81) and when samples were analyzed by product (breast: P = 0.75; thigh: P = 1.00). Oxygen sensors showed that DO values in the broth were around 6 ppm and O2 values in the head space were 14-16% throughout incubation. PFGE demonstrated high genomic similarity of isolates in the majority of the samples in which isolates were obtained from subsamples A and M. RISA and DGGE results showed a large variability in the bacterial populations that could be attributed to sample-to-sample variations and not enrichment conditions (aerobic or microaerobic). These data also suggested that current sampling protocols are not optimized to determine the true number of Campylobacter positive samples in retail boiler meat. Conclusions: Decreased DO in enrichment broths is naturally achieved. This simplified, cost-effective enrichment protocol with aerobic incubation could be incorporated into reference methods for the isolation of Campylobacter spp. from retail broiler meat.
isolation of Campylobacter spp. from clinical and food
samples has always been done using microaerobic
conditions, generally 85% N2, 10% CO2 and 5% O2, during
the enrichment of the samples and during the
incubation of plate media. Different methods have been
developed to generate microaerobic atmospheres and for a
small number of samples, sachets that generate CO2 are
commonly used . If a larger number of samples are
processed weekly, the evacuation-replacement is a more
economical alternative. In this system, the air in the jar
is partially removed by a vacuum pump and then
replaced with a microaerobic gas mix. For a large
number of samples, or to create unique microaerobic
gas mixes with increased H2 content, more sophisticated
microaerobic workstations have been developed .
Besides generating microaerobic conditions, several
O2-quenching agents have been traditionally added to
enrichment broths and agar plates for the isolation of
Campylobacter spp. These agents neutralize the toxic
effects of oxygen radicals and include blood or alkaline
hematin [8; 9], charcoal , iron salts and
norepinephrine , and ferrous sulfate, sodium metabisulfite
and sodium pyruvate (known as FBP supplement) .
In general, if blood or charcoal is added to agar plates,
no other O2 quenching compounds are added . To
ensure the microaerobic gas mix for the length of
incubation (at least 48 h) sealed jars are commonly used,
although plastic bags utilized to freeze food products
with a ziplock type closing to prevent air leaks have
been successfully used with gas-generating sachets and
manual evacuation-replacement systems [13; 14].
Although a microaerobic mix is indispensable to grow
Campylobacter spp. on agar plates, we have long
suspected that no extra addition of any microaerobic gas
mix is needed to keep Campylobacter spp. alive or even
grow them in enrichment broths. In the present study
we evaluated 108 retail broiler meat samples and
compared the efficacy of Bolton broth incubated under
microaerobic conditions using an
evacuation-replacement system (subsamples M) versus incubation under
aerobic conditions (subsamples A) for the isolation of
naturally occurring Campylobacter spp. Presumptive
Campylobacter spp. collected on agar plates were
confirmed and identified with multiplex polymerase chain
reaction (mPCR) assays and their DNA relatedness was
analyzed using pulsed-field gel electrophoresis (PFGE).
In addition, enriched broth cultures were analyzed with
ribosomal intergenic spacer analysis (RISA) and
denaturing gradient gel electrophoresis (DGGE) to determine
the variability in the total bacterial population profiles of
enrichment broths from subsamples M and A. Our
results indicate that microaerobic conditions that allow
Campylobacter spp. to grow are naturally created in
enrichment broths without the addition of extra
microaerobic gas mix, and therefore a simplified method has
been developed to identify these bacteria in food
Similar number of Campylobacter positive subsamples
From 108 retail broiler meat samples analyzed for the
presence of Campylobacter spp., 48 (42%) were
positive from the microaerobic subsamples (subsamples
M), and 46 (44%) were positive from the aerobic
subsamples (subsamples A). Combining the data from
subsamples M and A resulted in a total of 56 (52%)
positive samples for Campylobacter spp. Statistical
comparison by chi-square showed that the number of
Campylobacter positives from subsamples M and A
were similar (P > 0.05), even when analyzing the
subsamples by product (breasts or thighs) (Table 1). The
sensitivity, specificity and accuracy were high (0.78 or
above), and the Kappa values were above 0.50 for all
comparisons, with the observed agreement in the
Kappa value (considered the best agreement) always
above 0.7 . These high values reflected the large
number of samples that were either positive (38
samples) or negative (52 samples) in both subsamples M
and A, as calculated by 2-by-2 tables (data not shown).
Receiver operating characteristic (ROC) curves also
showed that the true positive fraction was high and
within the 95% confidence interval calculated for this
dataset (Figure 1).
mPCR assays identified both C. jejuni and C. coli species
Table 2 shows the number of isolates collected and
identified from subsamples M and A, and for each
product type. A 100% agreement was found between the
mPCR assay described in Materials and Methods and
the mPCR extensively used in our laboratories [16; 17].
All Campylobacter isolates were confirmed as either C.
jejuni or C. coli, with C. jejuni comprising 83% and 85%
of the isolates for subsamples A and M, respectively. In
32 samples, subsamples M and A had C. jejuni, while
six samples yielded C. coli in both subsamples. In 18
samples, only one of the subsamples (either M or A)
was positive for Campylobacter.
Table 1 Number of subsamples M and A that were
positive for Campylobacter spp.
a A chi-square values 3.84 assumes the null hypothesis that means from the
reference method (microaerobic conditions) are equivalent to means from the
test method (aerobic conditions) and cannot be rejected at the 5% level of
confidence (P < 0.05).
Figure 1 ROC curves. A high true positive fraction is shown with the upper and lower 95% confidence interval values. Consistent results were
obtained from subsamples M (microaerobic conditions) and subsamples A (aerobic conditions) indicating that both methods were equivalent to
isolate Campylobacter spp. from retail broiler meat.
PFGE similarity was high for most isolates collected from
subsamples M and A
PFGE analysis of 48 isolates (24 samples) showed a high
genomic DNA relatedness between strains from
subsamples M and the corresponding isolates from subsamples
A (Figure 2). For 14 isolates (7 samples), the similarity
between isolates from subsamples M and A was lower
than 90% (Figure 3).
Table 2 Speciation of Campylobacter isolates using the
mPCR assay described in Material and Methods and a
previously described mPCR assay .
Breast Thighs Breast Thighs
Bacterial diversity measured by RISA and DGGE studies
vary considerably among samples and subsamples
The results from the ARISA analysis of 41 subsamples
M and 41 complimentary subsamples A, chosen at
random, showed a large variation in the microbial
community and a lack of similarity patters intra- or
intersample (Figure 4). Similar results were found using
BioNumerics and the Pearson correlation to compare the
band patterns of subsamples M and A by DGGE. Even
when analyzing the data using the Dice coefficient,
which takes into account band migration, the results
from subsamples M and A showed low DNA similarity
at a cutoff point of 90% (data not shown). Table 3
shows the nearest neighbor identified from a BLASTn
comparison of DGGE band sequences from subsamples
M and A. Sequencing information suggested that the
bacteria present in most subsamples were facultative
anaerobes and microaerobic organisms. BLAST results
indicated a high degree of similarity of some rDNA
amplicons (> 90%) with Acinetobacter sp.,
Campylobacter jejuni, Lactobacillus sp. and Pseudomonas sp., and
lower identity (80-90%) with Lactobacillus sp. and
Figure 2 PFGE results. Isolates collected from subsamples M showing a high degree of similarity (> 90%) to isolates collected from subsample
A. Pairwise comparisons were done using the Dice correlation and clustering analyses with the unweighted pair group mathematical average
(UPGMA) clustering algorithm of BioNumerics ver. 5 (Applied Maths, Austin, TX, USA). The optimization tolerance was set at 2% and the position
tolerance for band analysis was set at 4%.
Figure 3 PFGE results. Isolates collected from subsamples M showing a low degree of similarity (< 90%) to isolates collected from subsample
A. Pairwise comparisons and cluster analyses were done as described in Figure 2.
uncultured bacterial species. The sample that showed
the largest bands separation had five well-distributed
DNA bands in the gel (Table 3 A through E; DGGE
bands K1 through K5). This sample was used
consistently in DGGE gels as marker to normalize the gels
and to allow for gel-to-gel comparisons using
BioNumerics. A BLAST comparison showed that the
sequences from these bands were similar to
Acinetobacter sp. and Lactobacillus sp. (Table 3).
O2 content decreased during the incubation of
In samples incubated in Bolton broth without the
addition of any microaerobic gas mix, the amount of O2 in
the head space of the bags decreased over time and was
at or below 17% at 24 h of incubation. The amount of
O2 in the atmosphere was stable between 14 and 16%
by 30 h of incubation; however, the amount of O2 never
reached less than 14% (Figure 5). The amount of
dissolved O2 in the enrichment broth, measured one inch
from the bottom of the enrichment bags, reached 6
ppm at around 6 h of incubation. This value was stable
thereafter and never reached above 7.5 ppm (Figure 6).
The presence of naturally occurring Campylobacter spp.,
either C. jejuni or C. coli, did not alter any of the values
obtained with the sensors. In addition, incubation of 100
ml of Bolton broth without meat samples and without
the addition of blood resulted in a similar pattern of
DO values. In samples in which the O2 sensors were
double bagged and gassed with a microaerobic gas mix,
the DO decreased to around 5 ppm and remained stable
for up to 72 h (data not shown). Identical patterns of
dissolved O2 levels were found when using ziplock
plastic bags commonly used to freeze food products (The
Glad Products Company, Oakland, CA) (data not
Several methods have been developed to generate
microaerobic conditions for the growth and
multiplication of Campylobacter spp. These methods are routine
and are consistently used during the enrichment of food
samples or during the incubation of inoculated plate
media. However, little is known about the actual
changes in O2 content in enrichment broth media
during incubation (37C or 42C). Our experiments were
aimed at determining the changes of O2 content in the
broth and in the air of the head space of the bags used
to enrich the samples for the isolation of Campylobacter
from retail broiler meat. The premises of this work was
that the incubation of enrichment broth may naturally
create microaerobiosis conducive to the grow of
Campylobacter spp. Samples were therefore divided in two
subsamples which were in turn incubated under
microaerobic conditions (M) or aerobic conditions (A).
We used an unpaired sample design, where the
enrichment conditions differ between the reference
(subsamples M) and the alternative method (subsamples
Figure 4 Results from RISA analysis. A low percentage of DNA similarity was found between the DNA profiles from subsamples M and the
DNA profiles from subsamples A.
Table 3 Results from BLAST analysis of sequenced DGGE bands.
Marker bands were used in all the gels.
a Unique DGGE bands from each subsample.
BLAST nearest homology
(GenBank accession number)
Figure 5 Oxygen measurements. Percentage of O2 in the head space of plastic bags throughout 48 h of incubation at 42C. Average SEM
of six measurements from subsamples positive for Campylobacter spp. after incubation under aerobic conditions. Measures were taken with an
O2 sensor (Vernier, Beaverton, OR) as the percentage of O2 in the air in the head space.
Figure 6 Oxygen measurements. Amount of dissolved oxygen (DO) in ppm in the enrichment broth. DO was measured at 1 inch from
bottom of the bags, throughout 48 h of incubation at 42C. Average SEM of six measurements from subsamples positive for Campylobacter
spp. after incubation under aerobic conditions. Measurements were taken with a dissolved oxygen sensor (Vernier) and amount of oxygen in the
liquid was recorded as mg/l or ppm.
A), and confirmed all presumptive positives using the
same molecular protocols. Because the comparison of
two qualitative methods is best accomplished near the
limit of detection of these methods, we used naturally
contaminated broiler meat samples, which have the
lowest contamination that can be naturally found [4; 17].
The statistical analyses of data from unpaired samples
are performed in the same way as for paired samples,
mainly using McNemars chi square test . The
number of Campylobacter positive subsamples was
statistically similar between subsamples M and A, and all
isolates were clearly identified as C. jejuni or C. coli.
These results demonstrate that enrichment broths
incubated under normal, aerobic conditions are sufficient to
detect Campylobacter spp. in retail broiler meat. There
was an increase in number of total positive samples by
10% when combining the result of the two subsamples.
These findings have been already reported several times
for commercial broiler meat naturally contaminated
with Campylobacter spp. [4; 17]. In addition, a ROC
curve of the data showed a high true positive fraction,
or rate, and a very low false positive fraction, which
indicated a very strong correspondence in the results
between the reference (subsamples M) and the
alternative methods (subsamples A).
The traditional methodology of enriching 25 g of meat
is the one suggested by the Bacteriological Analytical
Manual of the Food and Drug Administration (FDA)
, the Microbiology Laboratory Guidebook of the
Food Safety and Inspection Services of the U. S.
Department of Agriculture (FSIS UDSA) , the International
Organization for Standardization , the Health
Protection Agency of the UK , and several other
countries regulatory agencies. However, this methodology
does not appear to be optimized to detect the true
prevalence of Campylobacter spp. in retail broiler meat.
PCR analysis of the isolates showed that C. jejuni or C.
coli species are the only Campylobacter spp. found in
retail broiler meat. Some samples can be contaminated
with both species  but again the current
methodology used in food samples is not accurate enough to
reveal the extent of contamination of the same product
with different Campylobacter strains. PFGE analysis
further demonstrated that a single meat sample could
be contaminated with two, or maybe more, isolates from
the same species. For all practical purposes, C. jejuni
and C. coli are the only two Campylobacter spp. found
in retail poultry meat because no C. lari has been
identified since the introduction of molecular techniques for
routine identification of Campylobacter isolates,
approximately 15 years ago . The data collected with
the O2 sensors showed that the amount of O2 in the
enrichment broth was stable around 5-7 ppm after 6 h
of enrichment. These O2 levels can be obtained by
pressing out the air before closing the sample bags, and
without the need of any vacuum, as is required when
removing the air from a hard container. Whirl-Pak or
ziplock bags performed similarly, showing that they are
impervious to changes in the air trapped inside .
The fact that bags with only the enrichment broth
(without meat or blood) created microaerobic conditions
has encouraged us to continue this line of research, and
we are currently testing other broths without blood to
isolate Campylobacter spp. from retail broiler meat.
Therefore, an inexpensive, simplified method can be
developed for routinely use in the isolation and
detection of Campylobacter spp. from food products.
Incubation of broth under normal aerobic conditions,
with or without airspace, was done in the early 1980s to
isolate Campylobacter spp. from fecal samples , and
the use of 10% O2, 10% CO2 and 80% of N2 facilitated
and sustained the growth of Campylobacter spp. .
The ISO normative 10272-1:2006 requires a
microaerobic environment but provides for an alternative
incubation in a microaerobic atmosphere created by
screwcapped bottles or flasks filled with enrichment broth,
leaving a headspace of less than 2 cm, and tightly
closing the caps . But much has been speculated about
the need to have a higher surface area of the meat
samples during enrichment to yield a higher number of
positive samples under microaerobic conditions , or
the exact depth of the airspace, the appropriate ratio of
air to broth , and the correct type of incubation
container to promote the growth of Campylobacter
jejuni  to avoid significant difference in the results if
a microaerobic atmosphere is not used . Therefore,
the microaerobic conditions are routinely used to isolate
Campylobacter spp. However, our results do not suggest
any correlation between surface and microaerobic
conditions and do not support the notion that air to broth
ratio and the type of container are indispensable to
isolate Campylobacter spp. Our results point to the simple
fact that any closed plastic bag naturally produces
microaerobic environments conducive to the growth of
Campylobacter spp. without the need to add any
microaerobic gas mix. In our experiments, bags were closed
to leave a minimum airspace and the samples were
mixed, without stomaching, for few seconds. Thus, bags
with subsamples M had the same contact surface as
bags with subsamples A.
The microbial population of the enriched samples in
Bolton broth, as assessed by RISA and DGGE, was
diverse. There are no current data on the microbial
assemblage of retail broiler meat as a predictor to the
presence of a bacterial pathogen, such as
Campylobacter. Most of the work on the bacterial community of
broiler meat was done more than 20 years ago using
direct bacterial counts, and very few research studies
have used culture-independent methods to study the
microbial profile of these foods . It is known,
however, that some cold-tolerant bacteria, such as
Enterobacteriaceae, Acinetobacter and Pseudomonas, are
commonly present on broiler meat . These bacteria
are primarily facultative anaerobes or microaerobic
organisms, and the ribosomal RNA gene sequences
recovered in our samples, especially form the most
prominent bands from DGGE gels, had a high similarity to
these bacterial groups.
RISA and DGGE can be used to broadly characterize
the total microbial population in complex samples. The
results from these techniques were analyzed using the
Pearson correlation, which is the standard procedure for
comparison of densitometric curves [31; 32]. We
analyzed the results with the Pearson correlation and also
the Dice coefficient, which takes into account only the
band position and not the band thickness, as it is the
case in densitometric curves. Although the Dice
correlation showed a higher DNA relatedness among
corresponding M and A subsamples, the variability in the
bacterial populations in each set of subsamples was still
large and appeared to be more attributable to the
original bacterial composition of the sampled meat itself
than to the enrichment conditions (aerobic vs.
microaerobic). A significant limitation of DGGE-derived
phylogenetic data with the primers used in this study is the
relatively short rDNA sequence obtained from each
amplicon, thereby reducing the degree of phylogenetic
inference that may be assigned to each band. Yet, both
RISA and DGGE produced consistent results regarding
the variability in the bacterial assemblages associated
with retail broiler meat samples.
In summary, our results indicated that the enrichment
of retail broiler meat at 42C in closed plastic bags and
without the addition of a microaerobic mix is adequate
for the isolation of Campylobacter spp. With the
advancement of DNA-based biosensors and automation
for bacterial detection, enrichment broths could be
screened for the presence of Campylobacter spp. in a
shorter time, with greater sensitivity and without the
generation of any microaerobic condition. In addition,
food microbiology laboratories interested in establishing
techniques for the isolation of Campylobacter from
retail meat will have access to a cost-effective
enrichment procedure without the need to invest in systems
to generate microaerobiosis. Reference documents from
the FDA and FSIS USDA should eventually be updated
to provide for an alternative, simplified protocol that
yields similar number of Campylobacter positive samples
as the current reference protocols.
Sample preparation, incubation and Campylobacter
Retail broiler meat samples (total = 108 samples; 49
breasts and 59 thighs) were purchased from local stores
(Auburn, AL) from April 2009 to October 2010.
Samples were tested in batches of three to five samples per
week. Each meat package was considered one sample,
and from each package ~1-inch pieces were cut
aseptically and mixed thoroughly. For all samples, 25 g of
meat was weighed two times (two subsamples) in
individual, sterile Whirl-Pak (Nasco, Fort Atkinson, WI).
Each subsample was enriched in 100 ml of Boltons
broth (with antimicrobial supplements) and 5% (v/v) of
lysed horse blood . The control subsamples
(microaerobic subsamples) were incubated in anaerobic jars
gassed with a microaerobic gas mix (85% N2, 10% CO2,
5% O2; Airgas, Radnor, PA) using the
evacuation-replacement system MACSmics Jar Gassing System
(Microbiology International, Frederick, MD). The other
subsamples (aerobic subsamples) were incubated
without the addition of microaerobic gas mix, by closing the
bags after removing the remaining air manually. All
subsamples were incubated at 42C for 48 h.
After incubation and for all subsamples, 0.1 ml of the
enriched broth was transferred to modified charcoal
cefoperazone deoxycholate agar  through a 0.65 m
membrane filter as described elsewhere . All agar
plates were incubated under microaerobic conditions at
42C for 48 h. Presumptive Campylobacter colonies
were observed under phase contrast microscopy
(Olympus BX51, Olympus America Inc., Center Valley, P) for
spiral morphology and darting motility. Presumptive
isolates were stored at -80C in tryptic soy broth (Difco,
Detroit, MI) supplemented with 20% glycerol (v/v) and
5% (v/v) lysed horse blood for further analysis.
Identification of presumptive Campylobacter isolates by
Campylobacter isolates were recovered from frozen stocks
by transferring to Brucella agar plates supplemented with
5% horse blood and through 0.6 m membrane filters as
described above. Plates were incubated at 42C under
microaerobic conditions for 24 h. Bacterial DNA was
extracted using the Wizard Genomic DNA Purification
Kit as described by the manufacturer (Promega, Madison,
WI), but bypassing the RNA digestion step. Isolates were
identified with a previously described mPCR assay [17; 34;
35], and a newly developed mPCR comprised of two sets
of primers, one targeting the glyA gene of C. jejuni and the
other targeting the ask gene of C. coli. Gene sequences
downloaded from NCBI GenBank were aligned and
analyzed using Molecular Evolutionary Genetics Analysis
(MEGA) software  and primers were designed with the
Integrated DNA Technologies PrimerQuest software.
(Integrated DNA Technologies http://www.idtdna.com)
The sequences of the primers are shown in Table 4. C.
jejuni ATCC (American Type Culture Collection) 700819
and C. coli ATCC 43473 were used as control strains to
set up the PCR conditions. The annealing temperatures of
these primers were optimized with a gradient PCR
program of a DNA ENgine Thermal Cycler (Bio Rad
laboratories, Hercules, CA), and the final conditions for this
mPCR assay were 20 cycles of 94C for 30 seconds; 63C
for 1 minute and 72C for 1 minute. Amplified products
were detected by standard gel electrophoresis in 1.5%
agarose (Ultra Pure DNA Grade Agarose, Bio-Rad
Laboratories) in tris-borate-EDTA buffer at 100 V for 40 minutes.
DNA bands in the gels were stained with ethidium
bromide and visualized using a VersaDoc Imaging System
Typing of Campylobacter isolates with PFGE
Isolates from 31 samples for which both subsamples were
positive were randomly selected for PFGE analysis.
Campylobacter isolates were typed using pulsed-filed gel
electrophoresis (PFGE) following previously described
protocols [16; 23]. Briefly, DNA was digested with SmaI
and separated using a CHEF DR II system (Bio-Rad
Laboratories, Hercules, CA) on 1% agarose gels (SeaKem
Gold agarose; Lonza). The DNA size marker used in the
gels was Salmonella enterica subsp. enterica serovar
Braenderup strain H9812 (ATCC BAA-664) restricted
with XbaI. Restriction enzymes were purchased from New
England BioLabs (Ipswich, MA). Gels were stained and
visualized as described above (mPCR assays) and TIFF
images were loaded into BioNumerics version 6 (Applied
Maths, Austin, TX) for analysis. Pairwise-comparisons
were done with the Dice correlation coefficient, and
cluster analyses were performed with the unweighted pair
group mathematical average (UPGMA) clustering
algorithm. The optimization and position tolerance for band
analysis were set at 2 and 4%, respectively, and similarity
among PFGE restriction patters was set at 90%.
DNA extraction from enrichment broths for bacterial
DNA from enrichment broths after 48 h of incubation
(subsamples M and A) was extracted using the Wizard
Genomic DNA Purification Kit (Promega). To
determine the microbial community profile of these
subsamples, ribosomal intergenic spacer analysis (RISA) and
denaturing gradient gel electrophoresis were performed
Forty-one sample sets chosen at random (22 negative
for Campylobacter spp. in both subsamples and 19
positive for Campylobacter spp. in both samples [16 C.
jejuni/C. jejuni and 3 C. coli/C. coli]) were analyzed by
ARISA. RISA was generated by amplification of the
internal spacer region (ISR) using the universal primers
according to Cardinale et al. . Amplified products
were separated by electrophoresis on the NEN Global
Edition IR2 DNA Analyzer (LI-COR, Lincoln, NE)
following manufacturers instructions. RISA images were
processed with BioNumerics (Applied Maths). Following
conversion, normalization, and background subtraction
with mathematical algorithms, levels of similarity
between fingerprints were calculated with the Pearson
product-moment correlation coefficient (r). Cluster
analysis was performed using the UPGMA algorithm.
DGGE was performed using universal primers 338F
(containing a 5 G+C clamp) and 518R, which amplify a
segment of the 16S rDNA gene [38; 39]. PCR
amplification consisted of 30 cycles of 5 min of denaturation at
94C, 1 min of annealing at 55C, and 1 min of
extension at 72C. The DGGE system (Ingeny phorU,
Netherlands) had a denaturing gradient comprised of urea and
formamide ranging from 45% to 65% in vertical
polyacrylamide gels. Gels were stained with ethidium bromide
and visualized under a UV gel imager. As a standard
marker for gel comparison, every DGGE gel had one
lane containing a DNA marker that had five specific
bands. DGGE banding patterns were analyzed using
BioNumerics (Applied Maths). Pairwise comparisons and
cluster analysis were performed with the Pearson
correlation coefficient and the Dice coefficient, and the
UPGMA algorithm, respectively. The band position
tolerance was set at 3% and a cut off value of 90% was
used to determine similarity between subsamples.
Selected bands from DGGE gels were excised and
amplified using primers 338F (without the G+C clamp)
and 518R. Amplicons were purified using the Wizard
SV Gel and PCR Clean-up System (Promega), and PCR
products were sequenced with an ABI 3730 sequencer
(Applied Biosystems, Foster City, CA) at Lucigen
Table 4 Primers developed in this study for the specific identification of C.jejuni and C. coli.
TGGCGGACATTTAACTCATGGTGC CCTGCCACAACAAGACCTGCAATA GGCTCCTTTAATGGCCGCAAGATT AGACTATCGTCGCGTGATTTAGCG
Corporation (Middleton, WI). Sequences were aligned
with MultAlin  and the consensus sequences were
compared to the GenBank database using BLAST http://
blast.ncbi.nlm.nih.gov/Blast.cgi. The accession numbers
of the sequences deposited in GenBank are GU250527
Detection of O2 changes during the incubation of
The changes in the amount of O2 in the enrichment
broth and the head space in the enrichment bags was
measured in eight aerobic subsamples using a dissolved
oxygen (DO) sensor (amount of oxygen in liquid
measured as mg/l or ppm), and an oxygen (O2) sensor
(percentage of oxygen in the air). These sensors were
purchased from Vernier (Beaverton, OR). A double
bagging system was used to avoid air leaks during the
measurements taken with the O2 sensors during incubation.
Changes in O2 concentration were measured in all
subsamples. The O2 Gas Sensor was calibrated to the
environment within the plastic bag which produces
condensation (100% humidity), and therefore was started
at 20.1 O2 in percentage by volume. The DO sensor was
positioned in the enrichment bag with the collection tip
of the sensor placed at the bottom of the enrichment
broth with the subsample. The O2 sensor was placed in
the head space of the bag above the liquid. The excess
air was expelled from the bag before sealing and
incubation for 48 h. The DO sensor was calibrated by
prewarming the probe for 10 min in the broth before
starting the readings. Throughout incubation, the sensors
were connected to a laptop computer with the Logger
Lite data collection program (version 1.4) that
recorded readings every 1 min. The data were analyzed
using Microsoft Excel (Microsoft Corporation,
An unpaired sample design was used where the number
of Campylobacter positive subsamples enriched under
microaerobic conditions (reference method) was
compared to the number of Campylobacter positive
subsamples enriched under aerobic conditions (alternative
method). Statistical comparisons were made using the
formula mcnemar. test (x, y, correct = TRUE) of R ,
which is the McNemars chi-squared (g2) test for count
data, and it is based on McNemars Test for correlated
proportions . The accuracy, sensitivity, specificity,
and Kappa values for the test were calculated using
2by-2 tables according to Hanrahan and Madupu . A
receiver operating characteristic (ROC) curve was
determined with a web-based calculator with an ordinal
rating scale of 1 through 4, where 1 represents samples
that were negative for Campylobacter spp. in both
subsamples, and 4 represents samples that were positive
for both subsamples .
We thank Leslie Speegle for her assistance in collecting the sensor data and
Kennedy Wekesa for allowing us access to the phase contrast microscope.
JK work was supported by grant 0754966 from the Research Experiences for
Undergraduates Program of the Biology Directorate of the National Science
Foundation. The work of S.B. is supported by Science Foundation Ireland
PZ carried out the sample collection, the DNA preparation, PFGE and
PCRDGGE assays, and image statistical analysis. SKH helped with sample
collection and DGGE analysis. ML helped optimize the DGGE analysis. CRA
carried out RISA assays. SB helped analyze data and wrote part of the
manuscript. JRK carried out the primer design to differentiate C. jejuni from
C. coli. OAO conceived and coordinated the study, designed and revised the
manuscript. All authors read and accepted the final version of the
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