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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, multi-resistance 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
broiler farms.
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
DANMAP criteria.
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.
Introduction
Campylobacter 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 Campylobacter jejuni 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 isolates 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 flaA restriction fragment length polymorphism method.7 Multiple isolates from a farm were included, provided that the isolates showed Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus 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. aureus 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 Campylobacter 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 Enterobacteriaceae - >32 μg/mL. The breakpoints are shown in Table 1. The overall level of resistance to at least one antibiotic in the C. jejuni and C. coli 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 isolates were 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 isolates 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 C. jejuni 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 C. jejuni 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 Discussion
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 was at 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 Campylobacter 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. jejuni and 23.5% for C. coli),14 and Canada (22% for C. jejuni and 12% for C. coli).15 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 C. coli 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 isolates 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. jejuni 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. Acknowledgements
Financial support for this study was provided by the Rural Industries Research and Development Corporation (Chicken Meat Program). Transparency declarations
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 References
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Table 1. 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 Table 2. Results of MIC and disc diffusion tests for 125 C. jejuni and 27 C. coli isolates
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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