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Concurrent Quantitation of Total Campylobacter and Total Ciprofloxacin-Resistant Campylobacter Loads in Rinses from Retail Raw Chicken Carcasses from 2001 to 2003 by Direct Plating at 42°C

ABSTRACT This is the first report on the use of a normally lethal dose of ciprofloxacin in a Campylobacter agar medium to kill all ciprofloxacin-sensitive Campylobacter spp. but allow the selective isolation and quantitation of naturally occurring presumptive ciprofloxacin-resistant Campylobacter CFU in rinses from retail raw chicken carcasses (RTCC). Thermophilic-group total Campylobacter CFU and total ciprofloxacin-resistant Campylobacter CFU (irrespective of species) were concurrently quantified in rinses from RTCC by direct plating of centrifuged pellets from 10 or 50 ml out of 400-ml rinse subsamples concurrently on Campylobacter agar and ciprofloxacin-containing Campylobacter agar at 42°C (detection limit = 0.90 log10 CFU/carcass). For 2001, 2002, and 2003, countable Campylobacter CFU were recovered from 85%, 96%, and 57% of RTCC, while countable ciprofloxacin-resistant Campylobacter CFU were recovered from 60%, 59%, and 17.5% of RTCC, respectively. Total Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.52, 0.90 to 4.58, and 0.90 to 4.48 log10 CFU/carcass in 2001, 2002, and 2003, respectively. Total ciprofloxacin-resistant Campylobacter CFU loads in RTCC rinses ranged from 0.90 to 4.06, 0.90 to 3.95, and 0.90 to 3.04 log10 CFU/carcass in 2001, 2002, and 2003, respectively. Overall, total Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, 4 to 5 log10 CFU/carcass, respectively, were recovered from 16%, 32%, 26%, and 5% of RTCC tested over the 2-year sampling period. For the same period, total ciprofloxacin-resistant Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, and 4 to 5 log10 CFU/carcass, respectively, were recovered from 24%, 11%, 7%, and 0.2% of RTCC tested. There was a steady decline in total Campylobacter and total ciprofloxacin-resistant Campylobacter loads in RTCC rinses from 2001/2002 to 2003.

Many recent reports show that the usage of the fluoroquinolone group of antibiotics in poultry apparently creates a reservoir of ciprofloxacin-resistant Campylobacter jejuni and other Campylobacter spp. in the food chain in the United States (1, 4, 9, 10a, 21, 29, 31, 32). Recent surveillance data by the National Antimicrobial Resistance Monitoring Program (NARMS) clearly illustrate the emerging ciprofloxacin (a fluoroquinolone antibiotic) resistance of Campylobacter in humans (2,5,13). Molecular subtyping showed an association between ciprofloxacin-resistant C. jejuni from chicken products and acquired Campylobacter infections among Minnesota residents (29) who had contact with these products. Among Campylobacter spp., both C. jejuni and C. coli are recognized as predominant human-pathogenic species, showing the presence of ciprofloxacin-resistant strains, and these were frequently found in retail raw chicken carcasses (RTCC) (1,3,21,26). While ciprofloxacin resistance in Campylobacter can occur following the treatment of humans with the antibiotic, raw poultry is considered a significant source of ciprofloxacin-resistant Campylobacter (1,4,11,16,21,29,31,32). Therefore, there is a clear need for monitoring their persistence and quantitative reduction of the total ciprofloxacin-resistant Campylobacter load in the food chain, particularly from raw chicken products, in efforts to control human campylobacteriosis.
At the present time, the occurrence of ciprofloxacin-resistant C. jejuni or other Campylobacter spp. on raw chicken carcasses is determined by enrichment methods which provide only qualitative presence or absence tests per carcass sampled (12,13,26,34). Selective quantitative methods are yet to be developed for the quantitative monitoring of total ciprofloxacin-resistant C. jejuni and other Campylobacter loads persisting on raw and raw further-processed poultry products. The standard Campylobacter selective broth enrichment methods will not provide estimates of the original numbers of Campylobacter cells present per carcass. Also, culture isolation by broth enrichment techniques does not permit the total differentiated enumeration of the numbers or diversity of the ciprofloxacin-resistant versus ciprofloxacin-sensitive Campylobacter strains present in foods. The faster-growing strains will overgrow other strains. Genetic-based resistance to ciprofloxacin in human and animal isolates of Campylobacter has been established (7,14,31). A PCR-based TaqMan method was developed for the detection of C. jejuni isolates that carry the C3T transition in codon 86 of gyrA (24,33,35). However, there appears to be the involvement of multiple genes for resistance to ciprofloxacin (10,14), and a comprehensive set of PCR probes for these other loci has not yet been developed.
Confirmative tests based on complete gene sequencing or multiple PCR tests are indispensable for the complete characterization of ciprofloxacin-resistant Campylobacter. However, it is currently impractical by any DNA-based methods to isolate and quantitatively enumerate ciprofloxacin-resistant Campylobacter load from crude carcass rinses without destroying the Campylobacter cells present in those samples. Recently, a direct real-time PCR quantification of Campylobacter jejuni in chicken fecal and cecal samples has been published, but the minimum quantitation limit was 4 log 10 CFU/g (27). A culturebased direct plating method was recommended for the isolation and enumeration of total Campylobacter spp. from broilers (17,28), but no such method was published for ciprofloxacinresistant Campylobacter load. This is the first report of a direct plating method for selectively quantifying the persisting ciprofloxacin-resistant subpopulation of the total Campylobacter load on retail raw chicken carcasses.
Retail raw whole chicken carcass sampling source. From July 2001 through December 2003, individual commercially packaged refrigerated raw whole chicken carcasses (approximately 3 lb each) were sampled within their printed shelf dates generally at weekly intervals from local retail grocery stores in the Fayetteville, AR, area, at the rate of four carcasses per week. The samples were transported to the laboratory and sampled within 2 h of purchase.
Carcass rinse collection, subsample rinse concentration, and direct plating on CA and CCA. After removing from the retail package, each raw whole chicken carcass was placed in a 15-in. by 20-in. sterile poultry rinse bag (Nasco, Fort Atkinson, WI) and Butterfield's phosphate diluent (400 ml) was added to the bag. One-half of the Butterfield's phosphate diluent was poured into the interior cavity of the carcass and the other half on the outside of the carcass. To make sure all surface areas of each carcass were sampled, each carcass was rinsed inside and out with a rocking reciprocal motion in an 18-to 24-in. arc for 2 min. Then, the bag was aseptically cut at the lower corner to recover the whole carcass rinse into a sterile 500-ml bottle. The carcass rinse sample collected was mixed by gentle shaking prior to removing large subsamples for assay. Rinse subsamples were tested concurrently for each carcass for the determination of total Campylobacter counts and total ciprofloxacin-resistant Campylobacter counts by direct plating. Subsample volumes of 10 ml and 50 ml from the 400-ml rinse per carcass were concentrated by centrifugation at 8,000 ϫ g for 20 min, and the supernatant was carefully decanted without disturbing the pellet. Using all of the small volume (approximately Ͻ250 l) of rinse left in the tube after discarding the supernatant, the entire pellet was resuspended by pipetting and then direct plated on CA or CCA. Pellets obtained by centrifuging rinse subsamples (one 10 ml and one 50 ml for CA; both 50 ml for CCA) per carcass were direct plated on CA or CCA. CA and CCA plates were incubated under microaerophilic conditions in a Campy gas mixture (5% O 2 , 10% CO 2 , 85% N 2 ) at 42°C for 48 h. Typical presumptive Campylobacter microcolonies obtained from each rinse subsample concentrate on CA or CCA were enumerated as described below.
Campylobacter colony presumptive confirmation on CA and CCA, calculation of CFU counts, and statistical analysis of data. Characteristic total Campylobacter colonies on CA or CCA were presumptively identified after 48-h incu-bation at 42°C based on typical morphological characteristics (17,18,20,25). Typically, two types of Campylobacter colonies were found on CA or CCA after 48 h: (a) round, 1-to 2-mm-diameter, small, raised, smooth, shiny, convex, with a defined clear or translucent edge and a dirty brownish opaque center, and (b) flat, large, spreading with an irregular edge, clear, translucent, light cream, or grayish. Only a few non-Campylobacter contaminants grew on CA or CCA, and these were easily distinguishable from typical Campylobacter by their differences in colony morphologies. Representative characteristic Campylobacter colonies were examined by wet mounts for the presence of thin, curved, spiral cells with corkscrew motility. The representative presumptive Campylobacter colonies after their isolation on CA and CCA (a total of 16 Campylobacter isolates representing four carcasses per week) were randomly selected and preserved as frozen stocks after growth expansion. Genus and species identities for some selected Campylobacter isolates from CA and CCA were confirmed by PCR assays (19).
Total Campylobacter CFU recovered per carcass were calculated based on the following formulae: n 1 ϫ V 1 /V 2 or n 1 ϫ V 1 /V 3 , where n 1 is the total number of typical Campylobacter CFU recovered on CA at 42°C within 48 h per concentrated rinse subsample, V 1 is the total volume of chicken carcass rinse (400 ml), and V 2 and V 3 are the volume of subsample rinse used for concentration, 10 and 50 ml, respectively. Total ciprofloxacin-resistant Campylobacter CFU recovered per carcass were calculated based on the following formula: n 2 ϫ V 1 /V 3 , where n 2 is the total number of typical Campylobacter CFU recovered on CCA at 42°C within 48 h per concentrated rinse subsample and V 1 and V 3 are as described above. All Campylobacter count data were transformed to log 10 CFU/carcass using the Microsoft Excel program (Microsoft Corp., Redmond, Wash.). Mean log 10 CFU/carcass of total Campylobacter, mean log 10 CFU/carcass of total ciprofloxacin-resistant Campylobacter, standard deviations, correlation, and regression analyses were calculated using the Excel program.
Ciprofloxacin resistance confirmation of random Campylobacter colony picks on CA and CCA. Representative presumptive characteristic Campylobacter colonies randomly picked on CA or CCA (total of 16 new Campylobacter isolates representing four carcasses weekly) were spotted as individual spots (0.5-cm diameter) to generate fresh spiral cell growth; cell suspension (approximately 10 7 CFU/ml) was prepared by lifting each spot into 1 ml of Campylobacter enrichment broth (Bolton formula), each was then labeled as an individual Campylobacter isolate and then spotted (10 l per spot, approximately 0.5-cm diameter) onto a series of CCA plates containing different basal concentrations of ciprofloxacin (0, 2, 4, 8, 16, 32, 64, or 128 g/ml), and all plates were incubated at 42°C for 48 h under microaerophilic conditions to determine the ability of Campylobacter to grow at each ciprofloxacin concentration. Based on the agar dilution method according to NCCLS recommendations (23), a diverse set of C. jejuni and other Campylobacter spp. isolated on CA and CCA were tested concurrently for ciprofloxacin MICs on both CCA and Mueller-Hinton agar containing 5% defibrinated sheep blood, with C. jejuni ATCC 33560 as the quality control organism. The genetic basis for ciprofloxacin resistance of some selected Campylobacter isolates was confirmed by CampyMAMA PCR (36).

RESULTS AND DISCUSSION
One of the highest-priority research needs on Campylobacter was to develop laboratory methods for quantifying an antibiotic-resistant Campylobacter load persisting on raw poultry products to aid in risk assessment, to evaluate intervention strategies, and to develop meaningful baseline data for this pathogen. Currently, there is no published method for estimating loads of ciprofloxacin-resistant Campylobacter CFU within the total Campylobacter CFU load per chicken carcass. The recently published direct-plating method by U.S. Department of Agriculture (USDA)-Agricultural Resource Service (17,18) permitted the quantitative enumeration of Campylobacter CFU but not of antibiotic-resistant Campylobacter. Ge et al. (12) recently examined the antimicrobial susceptibilities of 378 Campylobacter species isolates obtained by an enrichment method from retail meats, but their method did not permit quantitation of the numbers of such antibiotic-resistant Campylobacter present in those meat products. Stern  Campylobacter spp. on processed broiler carcasses by a direct plating method but did not enumerate ciprofloxacin-resistant subpopulations present on those carcasses. Using the CA-or CCA-based direct-plating method, we determined quantitative trends in the numbers of total Campylobacter CFU (on CA medium) and total ciprofloxacin-resistant Campylobacter CFU (on CCA medium) at the rate of four carcasses per week by sampling a total of 420 carcasses in 105 weeks during the period from July 2001 through December 2003. Currently, there is no universally accepted best plating medium for isolating Campylobacter spp. In our direct-plating method, FDA-recommended Bolton formula was used in CA or CCA while Food Safety and Inspection Service and Agricultural Resource Service scientists adopted Campy-Cefex or modified campylobacter charcoal differential agar or Campy-Line agar media (17,25,28,30) for Campylobacter enumeration. We did not use NCCLS-recommended Mueller-Hinton agar supplemented with 5% defibrinated sheep blood because it was not recommended for the isolation of Campylobacter from crude carcass rinses. A normally lethal dose of ciprofloxacin in CCA permitted the direct isolation of primary colonies on CCA from a naturally occurring subpopulation of the ciprofloxacin-resistant Campylobacter cells if such cells are preexisting in crude chicken carcass rinses. The British Society for Antimicrobial Chemotherapy advised a minimum breakpoint of 2 g/ml for ciprofloxacin resistance in Campylobacter. Conversely, both Danish Veterinary and Food Administration and NARMS in the United States have adopted a breakpoint of 4 g/ml. In this study, we used a higher concentration of 8.6 g/ml ciprofloxacin in CCA medium (equivalent to 10 g/ml ciprofloxacin hydrochloride in CCA with 86% purity) due to the following criteria: (a) the lethal dose of 8.6 g/ml ciprofloxacin in CCA is greater than the 2ϫ ciprofloxacin breakpoint concentration of Յ4 g/ml used by NARMS and NCCLS for Campylobacter, which killed all ciprofloxacin-sensitive cells on CCA; (b) this lethal dose in CCA is lower than the minimum threshold limit of frequently found higher levels of ciprofloxacin resistance (MIC, Ն16 g/ml) in naturally occurring ciprofloxacin-resistant Campylobacter in chicken (12,22)  tween Ͼ4 and Յ8 g/ml), since such strains were rarely reported within the clinically significant ciprofloxacin-resistant Campylobacter isolated from chicken or human or other sources (12,22). For example, in a recent survey by Ge et al. (12) of 378 Campylobacter isolates from retail meats, 35% were ciprofloxacin resistant, but none of the resistant isolates had ciprofloxacin MICs between Ͼ4 and Յ8 g/ml but all had MICs of Ն16 g/ml. NARMS tested 297 Campylobacter isolates from humans, 288 Campylobacter isolates from chicken breast, and four Campylobacter isolates from ground turkey but reported that none of the resistant isolates had ciprofloxacin MICs between Ͼ4 and Յ8 g/ml but that all had MICs of Ն16 g/ml (22). Our direct-plating method accounted for the thermophilic group of total Campylobacter CFU on CA and total ciprofloxacin-resistant Campylobacter CFU on CCA, irrespective of species, and all recoverable at 42°C under microaerophilic conditions. The species-specific counts of different thermophilic campylobacters that may co-occur in crude carcass rinses, e.g., C. jejuni, C. coli, C. lari, or C. upsaliensis, were not determinable in this method. Using the recommended USDA-Food Safety and Inspection Service carcass rinse sampling procedure (25), this method permitted the detection and enumeration of a lower minimum level of total Campylobacter or total ciprofloxacin-resistant Campylobacter (about 8 CFU/carcass ϭ 0.90 log 10 CFU/carcass) due to the direct plating of pellets from centrifuged rinse subsample volumes of up to 50 ml from the total 400-ml rinse (equal to one-eighth of the total rinse volume), compared to the substantially higher minimum levels (about 1,000 to 4,000 CFU/carcass ϭ 3.0 to 3.6 log 10 CFU/carcass) obtainable by the direct plating of 0.1-ml subsample rinses of the total 100-or 400-ml rinse (equal to 1/1,000 or 1/4,000 of total rinse volume), as was used for Campylobacter enumeration by other researchers (17,18,25,30). Our direct-plating method, like those of Line et al. (17), Siragusa et al. (28), and Stern and Robach (30), determines only the numbers of Campylobacter CFU released from carcass skin into the rinse. Thus, this method does not reveal what numbers or percentages of total Campylobacter or total ciprofloxacin-resistant Campylobacter still remained firmly attached to carcass surfaces during the rinse sampling.
Total Campylobacter load in rinses from retail raw chicken carcasses from 2001 to 2003. Figure 1A shows the overall distribution of total Campylobacter loads in rinses from 420 RTCC sampled over a 2 1 ⁄2-year period. Figure 2A  Campylobacter loads of 0.90 to 2.0, 2 to 3, 3 to 4, and 4 to 5 log 10 CFU/carcass, respectively. Campylobacter incidence rates of 44% to 91% were frequently reported from retail raw chicken in the United States (6,26,34), but to the best of our knowledge, there are only a few reports about Campylobacter counts per carcasses at retail (20).
Total ciprofloxacin-resistant Campylobacter load in rinses from retail raw chicken carcasses from 2001 to 2003. Figure 1B shows the overall distribution of total ciprofloxacin-resistant Campylobacter loads in rinses from 420 RTCC sampled over a 2 1 ⁄2-year period. Figure 2B (12,13,26,34). None of the above other research reports gave numbers for total ciprofloxacinresistant Campylobacter CFU/carcass on samples they tested for Campylobacter.
In conclusion, our 2 1 ⁄2-year analysis of RTCC in one geographical area shows continuing persistence of countable numbers of total Campylobacter and total ciprofloxacin-resistant Campylobacter while there were some reductions in their incidence and loads from 2001/2002 to 2003. Random colony picks on CA and CCA confirmed the presence of subpopulations of ciprofloxacin-resistant C. jejuni (ciprofloxacin MICs ranging from Ն16 to Յ128 g/ml in both hippurate-positive and hippurate-negative strains) and of ciprofloxacin-resistant other Campylobacter spp. in RTCC rinses (data not shown), but their differential quantitation in carcass rinses must await further development of selective methods.