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Antibiotic resistance in Campylobacter jejuni and C. coli isolated
from poultry in the South East Queensland region
Jeanette K. Miflin, Jillian M. Templeton* and P. J. Blackall
Department of Primary Industries and Fisheries, Animal Research Institute,
Yeerongpilly, Queensland 4105, Australia
Key words: MIC, disc diffusion,
Running title:- Antibiotic resistance in Campylobacter
from Queensland poultry
*Corresponding author. Tel: +61-7-33629520; Fax: +61-7-33629429; E-mail:
Objectives: The aim of this study was to determine the antimicrobial resistance
patterns of 125 Campylobacter jejuni and 27 C. coli isolates from 39 Queensland
Methods: Two methods, a disc diffusion assay and an agar-based MIC assay,
were used. The disc diffusion was performed and interpreted as previously
described (Huymans and Turnidge Pathol 1997 29:209-216) while the MIC assay
was performed according to CLSI/NCCLS methods and interpreted using
Results: In both assays, no C. jejuni or C. coli isolates were resistant to
ciprofloxacin or chloramphenicol, no C. coli were resistant to naladixic acid and
no C. jejuni were resistant to erythromycin. In the MIC assay, no C. jejuni
isolate was resistant to naladixic acid while three isolates (2.4%) were resistant in
the disc assay. The highest levels of resistance of the C. jejuni isolates was
recorded for tetracycline (19.2% by MIC and 18.4% by disc) and ampicillin
(19.2% by MIC and 17.6% by disc). The C. coli isolates gave very similar results
(tetracycline resistance 14.8% by both MIC and disc; ampicillin resistance 7.4%
by MIC and 14.8% by disc).
Conclusions: This work has shown that the majority of C jejuni and C. coli
isolates were susceptible to the six antibiotics tested by both disc diffusion and
MIC methods. Disc diffusion represents a suitable alternative methodology to
agar-based MIC methods for poultry Campylobacter isolates.
is the most common bacterial cause of foodborne disease in
Australia.1 Contaminated animal products, particularly undercooked or raw poultry
meat and raw milk, are recognised as being the primary vehicles of human infections.2
While most cases of Campylobacter
infection are acute and self-limited in nature and
do not require antibiotic treatment,3 antibiotic treatment may be necessary in severe
cases or in immunocompromised patients.4
There is little Australian data on the levels of antibiotic resistance in animal
isolates of Campylobacter
. The only extensive prior Australian study5 used a disc
diffusion method to examine 213 poultry isolates.
We report on the antimicrobial susceptibility patterns present in 125
and 27 C. coli
isolates collected from 39 broiler farms in South-
Materials and methods
The 125 C. jejuni
and 27 C. coli
isolates used in this study were all confirmed by
PCR6 and were obtained during a large epidemiological study. The C. jejuni
came from 39 broiler farms while the C. coli
isolates came from 14 farms (all farms
being a subset of the 39 farms yielding the C. jejuni
isolates). All the isolates had
been genotyped by the fla
A restriction fragment length polymorphism method.7
Multiple isolates from a farm were included, provided that the isolates showed
ATCC 25922, Pseudomonas aeruginosa
ATCC 27853 and
ATCC 25923 were used as control strains.
All incubation of Campylobacter
species was performed at 37ºC in a modified
atmosphere incubator with a microaerobic atmosphere of 5% O2, 10% CO2 and 85%
N2. The other bacteria were incubated at 37 ºC in air.
Antimicrobial susceptibility testing – disc diffusion
The disc diffusion methodology was based on the National Committee for Clinical
Laboratory Standards.8 The disc content was as follows:- ampicillin 10 µg,
chloramphenicol 30 µg, ciprofloxacin 5 µg, erythromycin 15 µg, naladixic acid 30 µg
and tetracycline 30 µg. All discs were sourced from Oxoid. The isolates were grown
on brain heart infusion agar (Becton Dickinson #4311037) containing 5% sheep blood
cells (Bio Merieux #04378) at 37ºC for 48 h in the modified atmosphere incubator
described above. The NCCLS method8 was followed using a growth method
inoculum with the exception that the turbidity of the inoculum was adjusted to the
equivalent of a 1.0 McFarland Turbidity Standard. A purity check, performed by
inoculation onto sheep blood agar, was performed for all suspensions. The inoculated
Mueller Hinton Agar (MHA) with lysed horse blood (Oxoid #PP2097) and purity
check plates were incubated for 44-48 hrs at 37ºC in the modified atmosphere
incubator. For each test run, E. coli
ATCC 25922, P. aeruginosa
ATCC 27853 and S.
ATCC 25923 were used as control strains. The control strains were tested
using MHA (Oxoid #PP2096) plates incubated aerobically at 37 ºC. The results for
the control strains were read after 18-24 hours incubation. The interpretation of
susceptible, intermediate and resistant was based on the criteria of Huysmans and
Antimicrobial susceptibility testing – MIC testing
The MIC testing was done by a standardised agar dilution method8 with the exception
that the turbidity of the inoculum was adjusted to the equivalent of a 1.0 McFarland
Turbidity Standard. The inoculated MHA and purity check plates were incubated for
44-48 h at 37ºC in the modified atmosphere incubator. For each test run, control
strains (all three listed above) were used (as described above). The results for the
control strains were read after 18-24 hours incubation while the Campylobacter
results were read after incubation for 44-48 hours.
Antibiotics were tested in a two-fold concentration series – ampicillin (0.25 to
64 mg/L), chloramphenicol, ciprofloxacin and erythromycin (0.25 to 32 mg/L),
naladixic acid (1 to 128 mg/L) and tetracycline (0.25 to 128 mg/L). The presence of
growth was determined by visual examination and the MIC defined as the lowest
concentration of the antibiotic to prevent growth. Interpretation of the results of the
isolates was performed using the resistance breakpoints published by
DANMAP 2004.10 As DANMAP200410 does not contain a breakpoint for ampicillin,
we used the resistance break point used by the NCCLS for ampicillin resistance in
- >32 μg/mL.
The breakpoints are shown in Table 1.
The overall level of resistance to at least one antibiotic in the C. jejuni
isolates was compared by Chi-square analysis (Statistix Software).
The results of the disc diffusion and MIC testing are shown Table 2. At all times the
results from the control strains were within the range indicated as acceptable by the
There was strong agreement in the MIC and disc diffusion methods for all six
antibiotics tested for both Campylobacter
species (Table 2).
The level of resistance to any antibiotic examined in this study never exceeded
20% for either C. jejuni
or C. coli
. Amongst the 125 C. jejuni
isolates, the highest
level of resistance was to tetracycline (19.2% by MIC and 18.4% by disc) and
ampicillin (19.2% by MIC and 17.6% by disc). A similar level of resistance to these
same two antibiotics was found in the 27 C. coli
isolates tested (see Table 2). A low
level of resistance to naladixic acid (2.4%) was found in the C. jejuni
isolates by disc
while all the C. jejuni
isolates were sensitive by MIC. All C. coli
sensitive to this antibiotic by MIC and disc methods. A low level of resistance
(11.1% by MIC and disc) was found to erythromycin amongst the C. coli
while all C. jejuni
isolates were sensitive to this agent by both MIC and disc methods.
Resistance to more than one antibiotic was detected by disc diffusion in nine
isolates (7.2%) and by MIC in 11 C. jejuni
isolates (8.8%). All of these
isolates were resistant to both tetracycline and ampicillin. By disc diffusion and MIC
methods, none of the 27 C. coli
isolates showed resistance to more than one antibiotic.
The overall level of resistance (by both disc diffusion and MIC methods) was
not significantly different in C. jejuni
and C. coli
There were four broiler farms that contributed nine or more isolates of
to this study. In all of these cases, the genotyping indicated that – within the
farm – all the isolates tested were distinct and different genotypes. The occurrence of
resistance to ampicillin and tetracycline was not uniform within a farm – ranging from
11.1% to 30% of isolates (ampicillin) and from 18.2% to 30% of isolates
The on-going studies on the epidemiology of Campylobacter
in broilers in our
laboratory allowed the selection of isolates across a large number of broiler farms (39
in total). At the time these studies were performed, the number of broiler farms in the
South-East Queensland region was estimated to be 120. Hence, our study – based on
33% of the existing farms – provides a sound insight into the prevalence of
antimicrobial resistance in Campylobacter
associated with Queensland poultry. Our
selection of isolates was further guided by our knowledge, arising from the
epidemiological studies, of the different genotypes of C. jejuni /coli
present within a
flock. This knowledge of genotype allowed us to include multiple isolates from
within a flock – with the knowledge that each isolate represented a different genotype.
In contrast, the prior Australian study5 was based on isolates obtained from either
carcass rinses or intestinal samples – with no information on the genetic diversity or
The level of resistance we found to tetracycline for C. jejuni
and C. coli
the lower range of that reported in the prior Australian study (15-36%).5 Higher
levels of tetracycline resistance have been reported from four European Union
countries (35.4%)11 and the USA (43%).12 In Sweden, where tetracycline has not
been added to chicken feed since 1986,13 the level of tetracycline resistant
has been reported to be 1%.13
The level of resistance to ampicillin amongst both our C. jejuni
and C. coli
isolates was similar to that reported in other countries such as Germany (20% for C.
and 23.5% for C. coli
),14 and Canada (22% for C. jejuni
and 12% for C. coli
The prior Australian study has reported a much higher level of ampicillin resistance
The major difference between this study and the majority of similar studies
performed in other countries is the absence of resistance to ciprofloxacin. As
fluoroquinolones have not been registered for use in chickens in Australia, it was not
surprising to find that none of the 125 C. jejuni
and 27 C. coli
isolates were resistant
to this antibiotic. The prior Australian study has reported a similar absence of
ciprofloxacin resistance.5 In contrast, ciprofloxacin resistance has been reported in
the USA (19%),12 and a range of European nations (14.9% of C. jejuni
and 39.6% of
isolates).11 An absence or near absence of ciprofloxacin resistance has also
been reported from Brazil,16 Canada15 and Norway.17
In contrast to our findings, other studies have reported that C. coli
show higher levels of resistance than C. jejuni
isolates.11, 18 In a Northern Ireland
study that found no significant difference between the resistance patterns between C.
and C. coli
, the authors suggested that the uncommon occurrence of pig
husbandry on poultry farms in Northern Ireland might explain the lower rate of
resistance in C. coli
isolates.19 It is notable that none of the broiler farms represented
in our study involved co-location with pig husbandry operations.
Our finding of no multiple resistance (defined as resistance to four or more
different classes of antibiotics) has been also reported in a number of countries – four
European Union countries,11 Northern Ireland19 and Sweden.13
Our examination of multiple isolates (all genotypically different) within four
farms demonstrated that isolates within each farm could be both sensitive and
resistant to ampicillin and tetracycline, a finding that has been reported previously for
Overall, we found a good correlation between the disc diffusion methodology
of Huysmans and Turnidge9 and the MIC methodology. For those laboratories that
lack the capacity to undertake MIC based methodologies, disc diffusion represents, in
our view, an acceptable method for the determination of antimicrobial resistance
Our study has provided solid evidence that the majority of Queensland poultry
isolates of Campylobacter
show little resistance to antibiotics that are either used in
the poultry industry or which are of public health significance.
Financial support for this study was provided by the Rural Industries Research and
Development Corporation (Chicken Meat Program).
JMT currently leads a project funded by the Rural Industries Research and
Development Corporation (Chicken Meat Program) with some of the funds
employing a staff member of the laboratory. PJB is a member of the Chicken Meat
Research and Development Committee within the Rural Industries Research and
OzFoodNet Reported foodborne illness and gastroenteritis in Australia:
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Interpretation criteria used in this study. The disc diffusion criteria were
suggested by Huysmans and Turnidge9 while the MIC breakpoints, except where
Zone Diameter (mm) indicating Breakpoint
S = sensitive; I = Intermediate; R = resistant
b The figure in brackets is the concentration of antibiotic in the disc
c No breakpoint provided by DANMAP 2004.10 This is the breakpoint
provided by NCCLS8 for Enterobacteriaceae
Results of MIC and disc diffusion tests for 125 C. jejuni
and 27 C. coli
57.6 16.0 6.4 0.8 0 0 1.6 4.0 4.0 2.4 7.2 19.2
Vertical lines indicate breakpoints for resistance. The white fields denote dilution range tested for each antimicrobial. Values above the range
denote MIC values greater than the highest concentration in the range. MICs equal to or lower than the lowest concentration tested are given as
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