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Poult Sci 2008. 87:2152-2159. doi:10.3382/ps.2007-00441
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PROCESSING, PRODUCTS, AND FOOD SAFETY

Genotypic and Phenotypic Diversity Within Three Campylobacter Populations Isolated from Broiler Ceca and Carcasses

A. De Cesare*, A. Parisi{dagger}, V. Bondioli*, G. Normanno{ddagger} and G. Manfreda*,1

* Department of Food Science, Alma Mater Studiorum-University of Bologna, Via del Florio 2, 40064 Ozzano dell’Emilia (BO), Italy; {dagger} Experimental Zooprophilactic Institute of Apulia and Basilicata, 70017 Putignano (BA), Italy; and {ddagger} Department of Health and Animal Welfare, University of Bari, 70010 Valenzano (BA), Italy

1 Corresponding author: gmanfreda{at}disa.unibo.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The main aim of this study was to trace Campylobacter subtypes colonizing Italian broilers and carcasses in and between flocks. Overall, 209 Campylobacter isolates were collected from ceca (n = 94) and carcasses (n = 115) of broilers belonging to 3 different flocks reared in the same farm during subsequent rotations and processed in the same slaughterhouse. All isolates were identified by multiplex polymerase chain reaction and genotyped by amplified fragment length polymorphism. Furthermore, 166 out of 209 strains were phenotyped by antimicrobial resistance profile (R-type). The results of genetic and phenotypic characterization showed that (1) multiple Campylobacter species and subtypes can colonize the same broiler and carcass; (2) common Campylobacter subtypes in ceca and carcasses seem to be rare; and (3) carryover of Campylobacter subtypes between broiler flocks in the same house rarely occurs. The outcomes of this study should be taken into account for setting of isolate collection during epidemiological investigations to check sources and transmission routs of Campylobacter in broilers and poultry products.

Key Words: genotyping • phenotyping • Campylobacter • cecum • carcass


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The last summary report on trends and sources of zoonoses, zoonotic agents, and antimicrobial resistance in the European Union (http://www.efsa.eu.int/ science/monitoring_zoonoses/reports) showed that in 2005 there were 197,363 cases of campylobacteriosis in the 25 member states. The overall incidence of campylobacteriosis was 51.6 per 100,000, slightly higher than for Salmonella, making Campylobacter the most commonly reported gastrointestinal bacterial pathogen in the European Union. Campylobacter jejuni and Campylobacter coli occur in high numbers in the gastrointestinal tract of chickens (Rosef et al., 1985) and can be readily recovered from retail chicken products (Ge et al., 2003).

For this reason, handling of fresh meat and poultry products has been recognized as a primary risk factor in the transmission of campylobacteriosis in humans (Neimann et al., 2003).In Italy, Pezzotti et al. (2003) estimated a Campylobacter infection rate among broilers of 82.9% and a contamination frequency in chicken meat of 81.3%. Manfreda et al. (2006) quantified a mean Campylobacter load per broiler carcass around 5.16 log10 colony-forming units, identifying 96.7% of isolates colonizing carcasses as C. jejuni (49.2%) and C. coli (47.5%).

Data concerning the genetic diversity associated to Campylobacter strains colonizing both Italian broilers and poultry meat products are not currently available. The genetic subtypes of Campylobacter can be investigated by using several genotyping methods including PCR restriction fragment length polymorphism analysis of the flagellin gene (fla typing; Nachamkin et al., 1996), pulsed-field gel electrophoresis (PFGE; Ribot et al., 2001), amplified fragment length polymorphism (AFLP) analysis (Duim et al., 1999), automated ribotyping (Manfreda et al., 2003b), and multilocus sequence typing (Dingle et al., 2001). Comparisons among PFGE, fla gene typing, ribotyping, and AFLP for genotyping of thermotolerant Campylobacter suggested that AFLP is one of the most discriminatory methods (Lindstedt et al., 2000). A study comparing AFLP with multilocus sequence typing resulted in similar clustering of typed strains; therefore, both methods may be equally useful in disclosing genetic relationships for epidemiological studies (Schouls et al., 2003)

Genotyping data, showing similarities or differences among strains, should be supported by information concerning expression of phenotypic characters such as, for instance, antibiotic resistance. In this paper, antibiotic resistance profile (R-type) of Campylobacter isolates to ciprofloxacin, enrofloxacin, erythromycin, and tetracycline was investigated to support the classification of strains belonging to the same AFLP type within the same clone or strain type. The antimicrobials tested were those most commonly used in human enteritis treatments. In particular, erythromycin was the first macrolide to treat Campylobacter infections and it remains the treatment of choice for patients with uncomplicated enteritis in many countries (Skirrow and Blaser, 2000). Ciprofloxacin and enrofloxacin, other than for human treatment, are used in broiler medications (Gaunt and Piddock, 1996). Finally, tetracycline was used for many years for treatment of humans infected with C. jejuni and C. coli (Nachamkin et al., 2000)

In this study, AFLP and R-type were used to identify Campylobacter subtypes colonizing broiler ceca and carcasses. Then, presence of common subtypes between ceca and carcasses as well as persistence of specific subtypes in the same farm during different flock rotations were investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sampling

Three commercial broiler flocks, obtained from the same primary breeder and reared in the same farm, during consecutive flock rotations, were sampled in the same slaughterhouse in October 2003 (flock A), January 2004 (flock B), and April 2004 (flock C). Between flocks, the farm was cleared out; an all-in, all-out period of 2 wk was applied; and fumigation was performed before housing of the new flock

At the arrival to the slaughterhouse, all flocks were processed as first lot of the day. From each flock, cecal contents and carcass rinse waters from up to 10 birds were investigated. In particular, 5, 7, and 10 ceca and 9, 8, and 10 carcass rinse waters were tested within flocks A, B, and C, respectively.

Ceca were collected postevisceration and aseptically transferred to sterile bags chilled at 4°C. Carcasses were removed after the air-cooling operation and rinsed with 300 mL of sterile water. Bags were vigorously shaken by hand for approximately 1 min. After shaking was completed, carcasses were removed using sterile gloved hands, and rinses were aseptically transferred to smaller sterile bags chilled at 4°C. Both cecum and carcass rinse water refrigerated samples were transported to the laboratory within 3 h.

Bacteriological Analysis

Ceca were diluted 1:5 (vol/vol) using physiological water (0.85% NaCl) and homogenized in the PulsiFier (Microgen Bioproducts Ltd., Camberley, Surrey, UK) for 30 s before spread-plating of 0.1 mL onto Campy-Cefex agar plates (Stern and Line, 1992). Rinse water samples were serially diluted 1:10 in physiological water (0.85% NaCl) and plated onto the same selective agar plates. All inoculated plates were incubated at 42°C for 24 to 48 h in a microaerobic atmosphere (85% N2, 10% CO2, 5% O2), obtained by flushing the gas mixture through plastic zip-locking freezer bags, which were then hermetically sealed. After incubation, characteristic Campylobacter colonies were presumptively identified through phase-contrast microscopical visualization for typical morphological aspects and corkscrew movement. One to 5 Campylobacter-like colonies were isolated from each cecum and rinse water sample. All colonies were purified through 3 subcultures on tryptose blood agar base plates (Oxoid, Milan, Italy) supplemented with 5% laked horse blood (Oxoid) and incubated as described previously. All purified isolates were stored in Nutrient Broth n.2 (Oxoid) supplemented with 10% (vol/vol) glycerol (Difco, Becton Dickinson Italy, Milan) and incubated at –70°C until further characterizations.

Isolate Identification

Genomic DNA from Campylobacter isolates and positive as well negative control strains was extracted from 24-h tryptose blood agar base plate cultures suspended in 300 µL of 6% Chelex 100 resin (BioRad, Milan, Italy) according to Malorny et al. (2003).

Campylobacter jejuni and C. coli were identified using multiplex PCR as described elsewhere (Manfreda et al., 2003a). Amplification reactions were achieved using the following program: 1 cycle of 10 min at 95°C; 35 cycles of 30 s at 95°C, 90 s at 59°C, 1 min at 72°C; a final step of 10 min at 72°C. Expected PCR amplicons were at 857, 589, and 462 bp for genus Campylobacter, C. jejuni, and C. coli, respectively. All amplification reactions were achieved in a Mastercycler Gradient (Eppendorf, Milan, Italy). Positive control strains were represented by C. jejuni RM 1221 and C. coli RM 2228, whereas Staphylococcus aureus ATCC 51740 was used as negative control.

AFLP

All isolated and purified strains were analyzed by AFLP, according to the protocol published by Duim et al. (1999). Polymerase chain reaction was carried out on a 9700 GeneAmp PCR system (Applied Biosystems, Foster City, CA). The PCR products of each strain were separated on an ABI PRISM 310 genetic analyzer (Applied Biosystems) including Genescan ROX 500 (Applied Biosystems) as an internal size marker. The fragment size determination was performed using the Genescan 3.7 software (Applied Biosystems). The AFLP fragments detected in the size range between 50 and 500 bp were considered for numerical analysis. Genescan-processed data files containing bacterial AFLP profiles were imported into Bionumerics 4.61 software (Applied Maths, Saint-Martens-Latem, Belgium). Normalized AFLP profiles were compared using Dice correlation coefficient and clustered by unweighted pair group method with arithmetic mean. According to a previously published paper (Duim et al. 1999), a cut-off similarity value of 90% was fixed, and isolates whose AFLP patterns were >90% identical were assumed to be closely related genetically.

R-Type Determination

The isolate R-types were determined testing antimicrobial susceptibility to ciprofloxacin (0.25 to 8 µg/ mL; Bayer, Milan, Italy), enrofloxacin (0.12 to 4 µg/ mL; Bayer), erythromycin (0.25 to 32 µg/mL; Sigma, Milan, Italy), and tetracycline (1 to 32 µg/mL; Sigma) according to the guidelines of the National Committee for Clinical Laboratory Standards, document M-1-A2 (National Committee for Clinical Laboratory Standards, 2002). Minimum inhibitory concentration (MIC) determinations were performed by agar dilution method using Mueller-Hinton agar (Becton Dickinson) supplemented with 5% of defibrinated sheep blood (Oxoid). Minimum inhibitory concentration was defined as the lowest that completely inhibits colony formation. Standardized MIC breakpoints for antibiotic resistance are not available for Campylobacter. Therefore, the following MIC interpretative values were used as breakpoints: ciprofloxacin ≥4 µg/mL, enrofloxacin ≥2 µg/mL, erythromycin ≥8 µg/mL, and tetracycline ≥16 µg/mL. Quality control strains were C. jejuni ATCC 33560, Escherichia coli ATCC 25922, and Enterococcus faecalis ATCC 29212.

Statistical Analysis

Data collected were analyzed with Statgraphics package (version 5.1; StatSoft Inc., Tulsa, OK). Comparison between antibiotic resistance of C. jejuni vs. C. coli was performed using Fisher’s exact test. Moreover, the relationship between AFLP and R-type was tested by linear correlation evaluating the Pearson’s r coefficient. The value P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isolate Identifications

All 22 ceca and 27 carcass rinse water samples were Campylobacter positive by direct-plating onto Campy Cefex agar. A total of 209 Campylobacter strains were isolated from ceca and carcasses of broilers belonging to flocks A (n = 59), B (n = 53), and C (n = 97). In particular, 94 isolates were collected from ceca and 115 from carcass rinse waters. Overall, 155 (74.2%) isolates were identified as C. coli and 54 (25.8%) as C. jejuni by multiplex PCR. In particular, 66 (70.2%) cecum isolates were classified as C. coli and 28 (29.8%) as C. jejuni. Furthermore, 89 (77.4%) rinse water isolates were identified as C. coli and 26 (22.6%) as C. jejuni.

All cecum isolates from broilers belonging to flocks A and C were identified as C. coli, whereas those from flock B as both C. coli and C. jejuni. Furthermore, all carcass rinse water isolates from flock C were identified as C. coli, whereas those from flocks A and B were identified as both C. coli and C. jejuni. These last 2 species co-infected 33% of carcass rinse waters.

AFLP and R-types

All 209 Campylobacter strains isolated in this study were submitted to AFLP typing, whereas only 166 strains (i.e., 115 C. coli and 51 C. jejuni) were tested for R-type. Unfortunately, it was not possible to investigate R-type for all strains, because some of them were unable to grow after storage at –70°C.

A total of 15 different AFLP profiles (Figure 1Go) and 12 different R-types (Table 1Go) were identified among strains tested. The same AFLP profile was observed in 1 to 97 isolates and the same R-type in 1 to 68 isolates. The AFLP profiles associated to C. jejuni and C. coli were consistently different (Figure 1Go), whereas R-types G, L, M, S, and T were shared between both Campylobacter species. Furthermore, R-types D, E, H, and I were always associated to C. coli, whereas R-types B, C, and F were associated only to C. jejuni (Table 1Go).


Figure 1
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  		Figure 1. Amplified fragment length polymorphism (AFLP) profiles identified among Campylobacter isolates.

 

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  		Table 1. R-types identified among Campylobacter isolates
 
Table 2Go details identification, AFLP type, and R-type of isolates from broilers belonging to flock A. It was found that AFLP 9 was shared between 33 and 19% of cecum and rinse water strains, respectively. Moreover, cecum isolates were characterized by AFLP types 11 and 13, whereas rinse water strains were characterized by AFLP types 1, 2, 3, 10, and 12. All cecum strains showed R-type M, characterizing also 4.7% of rinse water isolates, presenting 5 additional R-types (i.e., F, B, C, S, and T; Table 2Go).


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  		Table 2. Genotypic and phenotypic profiles of isolates from flock A
 
In flock B, different AFLP profiles were identified among cecum (i.e., 4 and 5) and rinse water (i.e., 6, 8, 14, and 15) strains, whereas 3 (i.e., G, T, and L) out of 6 R-types, identified among cecum strains, were associated also to 83% of rinse water isolates (Table 3Go). Finally, in flock C, both cecum and rinse water strains showed the same AFLP profile, labeled as 7. Furthermore, both R-types identified among rinse water strains (i.e., I and D) were also associated to 92% of cecum isolates (Table 4Go).


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  		Table 3. Genotypic and phenotypic profiles of isolates from flock B
 

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  		Table 4. Genotypic and phenotypic profiles of isolates from flock C
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the United Kingdom, Sweden, the Netherlands, and Switzerland, studies suggest that conventionally reared broiler flocks are predominantly colonized with 1 or 2 Campylobacter subtypes (Jacobs-Reitsma et al., 1995; Berndtson et al., 1996; Shreeve et al., 2000; Ring et al., 2005), whereas in other countries, such as United States (Hiett et al., 2002), multiple Campylobacter subtypes are commonly isolated from broiler flocks. According to the results of this study, the Italian situation reflects that of United States. In fact, up to 3 different subtypes, characterized by different AFLP profiles belonging to a single species, were identified among cecum isolates of broilers belonging to the same flock. Moreover, up to 2 different Campylobacter subtypes were identified in the same cecum testing up to 5 colonies per sample. Concerning carcasses, 33.3% were co-infected by both C. jejuni and C. coli and 44.4% were contaminated by more than one AFLP type. In particular, up to 6 different AFLP types were detected among carcasses belonging to the same flock and up to 4 different AFLP types on the same carcass. According to Kramer et al. (2000), these results reinforce the epidemiological importance of selecting and testing more than one presumptive isolate per sample.

Overall, any significant correlation was observed between AFLP and R-types of the strains tested (P = 0.342) even if AFLP profile was significantly correlated to resistance to some antimicrobials, such as erythromycin (P = 0.04) and tetracycline (P = 0.03). Furthermore, a significant correlation was observed between AFLP and R-types for C. coli strains (P < 0.01), whereas for C. jejuni, a significant correlation was observed only between AFLP profile and resistance to tetracycline (P < 0.05). Campylobacter isolates with the same genetic profile, as determined by multilocus sequence typing and PFGE, but having different R-types have been described previously by other authors (Chu et al., 2004; Thakur and Gebreyes, 2005).

Studies on prevalence of Campylobacter spp. in poultry have shown that the species most frequently isolated is C. jejuni, whereas C. coli is less common and Campylobacter lari is rarely found (Tauxe, 1992). However, this, as well as other Italian studies (Pezzotti et al., 2003), showed that the most prevalent species colonizing Italian broilers seems to be C. coli. This might be due to the rearing conditions used in Italian conventional farms, where animals are reared up to 65 d, or to other management practices. The high incidence of C. coli in Italian broilers should be considered as a risk factor, because this pathogen shows increased resistance to a greater number of antimicrobials and it causes more indigenously acquired foodborne diseases than Salmonella enterica serovar Typhimurium (Tam et al., 2003).

A total of 76.5% of C. coli isolated in this study showed resistance to at least 1 antimicrobial, whereas 23.5% was sensitive to all molecules tested. In relation to C. jejuni, 47% was sensitive to all antibiotics, and 19.6 and 15.7% were resistant to fluoroquinolones or tetracycline, respectively. Tetracycline resistance did not show any statistically significant difference between C. coli and C. jejuni, whereas ciprofloxacin, enrofloxacin, and erythromycin resistance was significantly different among the 2 Campylobacter species (P < 0.001).

Unexpectedly, all 49 and 48 strains collected from cecum and rinse water samples in flock C showed the same AFLP type. Presence of a single subtype in broiler ceca belonging to the same flock might be due to its high colonizing potential (Ringoir and Korolik, 2003), whereas presence of a single strain on carcasses is unusual, because carcasses are in touch with many sources of cross contaminations. Possible explanations might be an extremely high adhesivity of AFLP type 7 strains to carcasses or an unintentional strain selection during the isolation process, even if it was performed avoiding enrichment (Newell et al., 2001). In fact, according to Miller et al. (2005), even use of direct-plating on media containing antibiotics might select for particular strains.

The AFLP types associated to cecum isolates from flocks A, B, and C were always different, even if birds were reared in the same farm. This result confirms observations of other authors, suggesting that, although carryover of infection might occur, it appears to be a minor source of colonization for subsequent broiler flocks in the same house (Shreeve et al., 2002). Moreover, it shows that routine broiler house cleaning or disinfecting procedures, or both, seem to be efficient to eliminate Campylobacter contamination.

Carcasses tested in this study were processed over a 3-mo interval, and this might explain why common strains were never identified among rinse water isolates collected over time. Strain-colonizing carcasses not identified in the ceca might come from different sources in the slaughterhouse and in the environment surrounding broiler houses as well as from contaminated crates during transport (Humphrey et al., 1994; Newell et al., 2001; Bull et al., 2006).

In conclusion, the results of this study show that (1) multiple Campylobacter species, characterized by different AFLP and R-types, colonize the same broiler and carcass; therefore, testing of more than 1 isolate per sample has to be taken into account to perform epidemiological studies; (2) the presence of the same Campylobacter subtype between cecum and carcass isolates seems to be rare; (3) carryover of Campylobacter subtypes between Italian broiler flocks in the same house rarely occurs. The outcomes of this study should be taken into account for the epidemiological investigations to check sources and transmission routs of Campylobacter in broilers and poultry products.


   ACKNOWLEDGMENTS
 
We would like to thank Alex Lucchi (Alma Mater Studiorum-University of Bologna) for help in determination of the R-types. This research was partially supported by the Italian Ministry of Health, grant IZS/ PB/03/2001.

Received for publication October 29, 2007. Accepted for publication May 11, 2008.


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 DISCUSSION
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