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Poult Sci 2008. 87:187-190. doi:10.3382/ps.2006-00407
© 2008 Poultry Science Association
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PROCESSING, PRODUCTS, AND FOOD SAFETY: Research Notes

Occurrence, Identification, and Characterization of Campylobacter Species Isolated from Portuguese Poultry Samples Collected from Retail Establishments

C. Mena*, D. Rodrigues*, J. Silva*, P. Gibbs{dagger} and P. Teixeira*,1

* Escola Superior de Biotecnologia, Rua Dr António Bernardino de Almeida, 4200-072 Porto, Portugal; and {dagger} Leatherhead Food International, Surrey, KT22 7RY, United Kingdom

1 Corresponding author: paula{at}esb.ucp.pt


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Campylobacter spp. are a leading cause of human diarrhea. The common source of infection is contaminated food, particularly poultry. From May 2005 to October 2006, 164 samples of poultry obtained from several retail establishments and from different Portuguese producers were analyzed for the presence of Campylobacter spp. A total of 99 Campylobacter strains were isolated and further identified. Susceptibilities for 44 strains were determined for 11 antimicrobial drugs by the agar diffusion technique. According to the minimal inhibitory concentration for 90% of the isolates, resistance to tetracycline, nalidixic acid, rifampicin, erythromycin, vancomycin and penicillin was observed. Sensitivity to gentamycin and chloramphenicol was also demonstrated. According to the antibiotic resistance profiles, the isolates appeared to differ from each other, including those that had been collected at the same period of time and from the same chicken sample.

Key Words: Campylobacter species occurrence • chicken • minimal inhibitory concentration


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The foodborne pathogen Campylobacter is a leading cause of gastrointestinal human infections in many industrialized countries, imposing a burden on public health (Friis et al., 2005; Keller and Perreten, 2006). The infective dose of Campylobacter is very low, which means that even a very small number of Campylobacter cells in food samples represents a potential health hazard (Liu et al., 2006). The infection is generally acute and self-limiting after a course of 3 to 5 d of severe gastroenteritis and abdominal pain, diarrhea, and dysentery with fecal blood and leukocytes (Liu et al., 2006); nevertheless, some severe cases might require antibiotic treatment. Campylobacter is also recognized as a major contributing factor to Guillain-Barré syndrome, the most common cause of acute paralysis in both children and adults (Funakoshi et al., 2006).

Campylobacter jejuni and Campylobacter coli together account for more than 95% of Campylobacter infections in humans (Oliveira et al., 2005), with C. jejuni being the predominant causative species (Snelling et al., 2005; Açik and Çetinkaya, 2006). Each species of Campylobacter has a favored reservoir; C. jejuni is predominantly associated with poultry and has evolved to preferentially colonize the chicken gut given its optimal conditions. Indeed, and although found in many farm animals, Campylobacter in poultry and poultry products is causing the most concern in many developed countries within the last 20 yr (Sahin et al., 2003; Snelling et al., 2005; Wittwer et al., 2005; Luber et al., 2006; Müller et al., 2006; Sherer et al., 2006; Rodrigo et al., 2007). The FAO/WHO (2004) risk assessment of Campylobacter reported that broiler chickens represent a model that includes all stages of the chicken production chain and can be used to generate baseline estimates of the risk of Campylobacter infection on per-serving bases.

A significant antibiotic resistance in Campylobacter spp. from human and animal sources has been reported (Aquino et al., 2002; Rodrigo et al., 2007). To our knowledge, and since 1992, there is no published report about prevalence and antibiotic resistance of Campylobacter spp. isolated from raw poultry meat in Portugal (Cabrita et al., 1992). The aims of the present study were as follows: i) to evaluate the occurrence of Campylobacter spp. in Portuguese food samples derived from poultry, ii) phenotyping (distinguish between C. jejuni and C. coli by using the hippurate test), and iii) to determine the susceptibility of Campylobacter spp. to 11 different antimicrobial agents.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Sampling
During May 2005 until October 2006, 164 samples of poultry were collected from different Portuguese producers and from several retail establishments located in the north of Portugal (Table 1Go). All the samples were transported to the laboratory under refrigerated conditions, and microbiological analyses were carried out immediately.


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  		Table 1. Occurrence of Campylobacter spp. isolated from Portuguese poultry samples during May 2005 to October 2006
 
Isolation and Identification of Campylobacter Isolates
According to the method for detection of Campylobacter spp. (ISO 10272-1; Anonymous, 2006), 25 g of poultry samples were homogenized in a stomacher (BagMixer 400, Interscience, St Nom, France) for 1 to 2 min in 225 mL of Bolton broth (Oxoid, Hampshire, UK) containing 5% of sterile lysed defibrinated horse blood (Oxoid) and then incubated under microaerophilic conditions (Gen-box microaer, bioMérieux, Marcy l’Etoile, France) at 37°C for 4 h and then at 41.5°C for 44 h. After incubation, 2 mL of the sample enrichment was heated in a water bath at 100°C for 15 min. The detection of Campylobacter was performed using the VIDAS Campylobacter method CAM (bioMérieux; Anonymous, 2004), which is an enzyme-linked fluorescent immunoassay performed in the automated VIDAS instrument using antibody specific for C. jejuni, C. coli, and Campylobacter lari; however, Campylobacter upsaliensis is not detected by this method. The strips of CAM VIDAS were inoculated with 0.5 mL of the heated sample, and the result was obtained automatically. Aliquots of enrichment cultures (unheated) of VIDAS-positive samples were streaked onto modified charcoal cefoperazone deoxycholate agar (Oxoid) and Campylosel agar (bioMérieux) or Campyfood agar (bioMérieux). Agar plates were incubated at 41.5°C for 48 h under microaerophilic conditions. Presumptive Campylobacter colonies were streaked to produce colonies on Columbia blood agar plates. The pure cultures were then confirmed according to the ISO 10272-1 (Anonymous, 2006) methods (i.e., gram-stained and tested for cell morphology, hippurate hydrolysis, growth in a microaerobic atmosphere at 41.5°C for 24 to 48 h, motility, and production of oxidase and catalase).

Antibiotic Susceptibility
Forty-four isolates were subjected to antimicrobial sensitivity tests using the agar dilution method described by the NCCLS (2004). The tested antimicrobial agents were as follows: ampicillin, penicillin, tetracycline, nalidixic acid, rifampicin, erythromycin, chloramphenicol, nitrofurantoin, ciprofloxacin, vancomycin, and gentamycin kindly supplied by Labesfal, Carnaxide, Portugal. Inocula were prepared of colonies from overnight growth on blood agar plates, by suspension in sterile Ringer’s solution to obtain a turbidity equivalent to 0.5 McFarland standard. Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212 were plated as controls.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Campylobacter spp. were detected in 99 out of the 164 samples analyzed (Table 1Go). Fourty-four isolates were arbitrarily selected for further phenotypic identification. According to the hippurate hydrolysis test, 68% were identified as C. jejuni and 32% as C. coli (data not shown). In fact, the higher occurrence of C. jejuni has already been associated with poultry samples (Luber et al., 2006; Rodrigo et al., 2007).

The occurrence of C. jejuni and C. coli in wild and farm animals has already been studied in Portugal (Cabrita et al., 1992); from 59 chicken samples, the occurrence was 60.2%; from 65 swine samples, the occurrence was 59.1%; from 31 black rats, the occurrence was 57.4%; from 61 sparrow samples, the occurrence was 45.5%; from 21 duck samples, the occurrence was 40.5%; from 32 cow samples, the occurrence was 19.5%; and from 27 sheep samples, the occurrence was 15.3%.

Table 2Go shows the minimal inhibitory concentration (µg/mL for 50 and 90% of the isolates,) of the isolates for the 11 antimicrobial agents tested. For the tested strains and according to the antibiotic resistance profiles, the isolates appeared to differ from each other, including those that had been collected at the same period of time and from the same chicken sample (data not shown). These agents were chosen based on those considered important for treating campylobacteriosis and for the surveillance needs of the National Antimicrobial Resistance Monitoring System (http://www.fda.gov/cvm/narms_pg.html#Data) for monitoring trends in susceptibility among agents used in human and veterinary medicine. According to the Campylobacter Sentinel Surveillance Scheme Collaborators, the study of Campylobacter infections as well as the antibiotic resistance profiles is of great importance, because this might explain the risk for acquiring antibiotic resistant strains [e.g., during foreign travel (Greig, 2003)]. Indeed, antibiotic resistance in Campylobacter is emerging globally and has already been described by several authors and recognized by the World Health Organization as a problem of public health importance (Greig, 2003; McDermott et al., 2005; Moore et al., 2006). Although comparable standardized procedures for susceptibility testing are available for a wide range of organisms, based on the guidelines defined by the NCCLS, no internationally accepted criteria are available for susceptibility testing of Campylobacter spp. As a result, there is a debate in the literature regarding the interpretation of the results of antimicrobial resistance (Ge et al., 2002; Moore et al., 2006).


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  		Table 2. Minimal inhibitory concentrations (MIC) for 50 and 90% of Campylobacter spp. isolated in Portugal during May 2005 to October 2006
 
Cabrita et al. (1992) reported the antimicrobial resistance of Campylobacter strains isolated from wild and domestic animals in the northeast of Portugal. These authors demonstrated that 5.5% of the strains were resistant to ampicillin, 5.5% to tetracycline, 12.6% to erythromycin, and 23.5% to streptomycin. Based on these results and as shown in Table 2Go, it is possible to conclude that a significantly higher resistance was observed in the Campylobacter isolates obtained from 2005 to 2006; the minimal inhibitory concentration for 90% of the isolates value for tetracycline was 512 µg/mL, for ampicillin was 128 µg/ mL, and for erythromycin was 256 µg/mL. In general, and for all the other tested antibiotics, the isolates were shown to be very resistant to the higher antibiotic concentrations. Nowadays, and based on these results, we may conclude that the overall antibiotic resistance to ampicillin, tetracycline, and erythromycin is significantly higher compared with the Campylobacter strains isolated previously (Cabrita et al., 1992). Corcoran et al. (2005) reported that the minimal inhibitory concentrations of erythromycin-resistant Campylobacter isolates ranged from 128 to >256 mg/mL; also, the study performed by Barton and Wilkins (2001) showed resistance of Campylobacter spp. isolates to ampicillin.

Campylobacter coli strains are most likely to be resistant to antibiotics and have been described as organisms that have a propensity to acquire resistance to macrolides, including erythromycin. In fact, and in contrast to C. jejuni, which largely remains sensitive to erythromycin, C. coli has frequently been found to be resistant to erythromycin and other macrolides (Cloak and Fratamico, 2002; Nayak et al., 2005; Kim et al., 2006). According to our results, this tendency was not observed (data not shown); the resistance and sensitivity of the strains was not correlated with the species.

Generally, the data recorded in the literature describes the sensitivity of Campylobacter spp. using the antimicrobial agents on discs with the exact concentrations of antibiotic (Aquino et al., 2002; Rodrigo et al., 2007); they describe zones of resistance and susceptibility to various antimicrobial agents. In this study, the susceptibility of some Portuguese food isolates has been evaluated by using the minimal inhibitory concentration method; thereby, the exact concentration in which the microorganism cannot grow and proliferate was determined. In fact, these values might be useful to estimate the risk of campylobacteriosis, because nowadays it is well-known that food, particularly poultry, is the principal vehicle of Campylobacter infection.


   ACKNOWLEDGMENTS
 
This work received financial support via European Project "Phagevet-P, Veterinary Phage Therapies as Alternatives to Antibiotics in Poultry Production," European Commission Specific Targeted Research Project in the Sixth Framework, FP-6-2003- Food-2-A, Food T1.2.

Received for publication November 28, 2006. Accepted for publication September 18, 2007.


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 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Anonymous. 2004. Vidas Campylobacter. bioMérieux, Marcy l’Etoile, France.

Anonymous. 2006. Microbiology of food and animal feeding stuffs—Horizontal method for detection and enumeration of Campylobacter spp. Detection method. International Standard ISO/10272-1.

Barton, M. D., and J. Wilkins. 2001. Antibiotic resistance in bacteria isolated from poultry. Rural Industries Research and Development Corporation Publication No. 1/105 Report of RIRDC Project No USA-9A.

Cabrita, J., J. Rodrigues, F. Bragança, C. Morgado, I. Pires, and A. P. Gonçalves. 1992. Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from Northeast Portugal. J. Appl. Bacteriol. 73:279–285.[Medline]

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