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The prevalence of Campylobacter upsaliensis currently may be underestimated due to the widespread use of unsuitable isolation procedures (2, 4). The poor growth of C. upsaliensis on modified cefoperazone charcoal deoxycholate (mCCDA), commonly used for the detection of other thermophilic campylobacters (7), has been attributed to its susceptibility to cefoperazone (2). Techniques based on membrane filtration are among the most efficient for isolating C. upsaliensis (1, 8, 12). However, filtration is costly and labor-intensive and may be unsuitable for samples containing small numbers of organisms. Although PCR-based (11, 13, 14, 17) and enzyme immunoassay-based (10) assays are promising, their roles for detecting C. upsaliensis have yet to be defined.

Using a modification of blood-free selective media containing reduced levels of cefoperazone, Aspinall et al. (2, 3) demonstrated recovery of C. upsaliensis on cefoperazone amphotericin teicoplanin selective medium (CAT) that was equivalent to recovery using membrane filtration and considerably superior to recovery on mCCDA. Subsequently, CAT was shown to be superior to mCCDA for the isolation of C. upsaliensis using a semiquantitative plating method (6). However, not all workers have found CAT to be ideal for recovering C. upsaliensis from clinical specimens (9), and the reasons for these contradictory results are unclear.

In a study aimed at identifying fresh clinical isolates of C. upsaliensis in stools of children and animals in Dublin, we noted that six C. upsaliensis isolates were recovered on CAT while none were recovered using incubation on mCCDA. To further investigate the reasons for the different yield of C. upsaliensis using these two regimens, we examined the antibiotic sensitivities of these six isolates. In addition, we characterized the effects of incubation time and antibiotic composition of media on the growth of a group of C. upsaliensis isolates obtained from the Laboratory Centre for Disease Control (LCDC), Winnipeg, Canada. Finally, we compared the recovery from spiked fecal samples of C. upsaliensis strains using these two media.

The bacteria used in this study comprised 11 C. upsaliensis strains obtained from the LCDC, 6 clinical isolates of C. upsaliensis obtained from human and animal stool specimens at Our Lady's Hospital, Crumlin, Ireland, and C. upsaliensis ATCC 43954. Thermophilic campylobacters were incubated at 37°C under microaerophilic conditions, generated using the CampyGen system (CN35; Oxoid Ltd., Basingstoke, Hampshire, England) to give approximately 6% oxygen and 10% carbon dioxide without evolution of hydrogen in anaerobic jars.

Media used included Columbia agar base (Oxoid CM331) containing 5% defibrinated horse blood and a Campylobacter blood-free selective agar base containing charcoal (Oxoid CM739). CCDA selective supplement (Oxoid), providing 32 mg of cefoperazone and 10 mg of amphotericin per liter, and CAT selective supplement (Oxoid), providing 8 mg of cefoperazone, 10 mg of amphotericin, and 4 mg of teicoplanin per liter, were added where appropriate. The in-house supplements amphotericin (Fungizone; Squibb) and teicoplanin (Targocid; Marion Merrell) were added to base agars, where indicated. MICs of cefoperazone were determined using the Etest (AB Biodisk, Solna Sweden). Brucella broth (Difco Laboratories, Detroit, Mich.) was used for suspension and dilution of organisms. McFarland tubes were used for standardization of inoculum (bioMerieux, Marcy l'Etoile, France). Stools used for spiking studies were first prescreened for enteric pathogens, including C. upsaliensis.

The original purpose of the screening study was to identify fresh clinical isolates of C. upsaliensis for pathogenicity studies. Over a 9-month period, a total of 2,064 stool specimens from hospitalized and nonhospitalized children with diarrhea were cultured on mCCDA and CAT agar. Stools were directly inoculated onto plates. mCCDA plates were incubated at 37°C in a microaerophilic atmosphere for 48 h in the clinical laboratory. The aim of the screening study was to achieve a maximum possible yield of C. upsaliensis. Therefore, CAT cultures were maintained for a longer incubation period of 96 h, as prolonged incubation time is associated with increased yield of organisms. The frequency of isolation of C. upsaliensis over the time period was retrospectively compared on both media. In addition, in order to increase the yield of isolates with which to work, rectal swabs from 24 asymptomatic animals (17 dogs, 4 cats, and 3 rabbits) attending a veterinary clinic were cultured on CAT agar and mCCDA for 96 h. As these samples were not processed by the clinical laboratory, the incubation period on mCCDA also was extended to 96 h. All Campylobacter species were fully identified using an API Campy system (API 20800; bioMerieux) or were identified at the Campylobacter Reference Laboratory, Public Health Laboratory Service, Central Public Health Laboratory, Colindale, United Kingdom.

Eleven strains of C. upsaliensis obtained from the LCDC and the type strain were cultured on Columbia blood agar without additives for 48 to 72 h at 37°C. Suspensions of each strain were made in brucella broth to a density equivalent to a number 6 McFarland standard. Ten microliters of each suspension were inoculated in duplicate on to CAT agar and mCCDA agar. This inoculum was streaked using a standard disposable 10-µl loop by the ecometric method described by Corry et al. (7). Briefly, the primary inoculum was spread using five parallel strokes, approximately 5 mm apart, over sector A. A fresh loop was used to repeat this pattern, at a 45° angle to sector A, forming sector B. This process was repeated three more times (sectors C through E). All plates were incubated in a microaeropilic atmosphere at 37°C, and growth was assessed ecometrically after 48, 72, and 96 h. The growth on each plate was expressed as the absolute growth index (AGI), depending on the number of streaks with visible growth. Growth on all five streaks (A through E) was designated an AGI of 5, growth on A through D was given an AGI of 4, growth on A through C was given an AGI of 3, and so on. A preliminary pilot study confirmed no appreciable effect on C. upsaliensis growth of repeated opening of gas jars compared with undisturbed incubation over 96 h (data not shown).

Etests with cefoperazone were performed on the six C. upsaliensis isolates recovered from stool samples during the screening study. Etests were performed in duplicate for each isolate. In order to examine the possibility that the composition of the agar base or the presence of teicoplanin could influence cefoperazone sensitivity results, each strain was subjected to Etests on blood agar base and charcoal base, both with and without 4 mg of teicoplanin per liter. Plates were dried for 30 min prior to use. Each set of plates was swab inoculated using the same suspension of organism in brucella broth at a 0.5 to 1.0 McFarland standard, according to the manufacturer's instructions for use with campylobacters. Plates were allowed to dry for 30 min at an ambient temperature prior to application of the strips. Plates were incubated in a microaerophilic environment, examined after 48, 72 and 96 h, and promptly returned to microaerophilic conditions.

The 11 LCDC C. upsaliensis isolates and the type strain used in ecometric plating studies were subjected to antibiotic disk sensitivity testing (30-µg cefoperazone disk). Isolates were tested in duplicate on Columbia blood agar and incubated under microaerophilic conditions.

The recovery from spiked human feces of C. upsaliensis on CAT and mCCDA was compared. Five strains of C. upsaliensis obtained from the LCDC that had been cultured on Columbia blood agar for 72 h were suspended in brucella broth to produce a turbidity equivalent to a number 6 McFarland standard. Serial 10-fold dilutions of each strain to extinction were made in brucella broth. One hundred microliters of each dilution was then added to 900 µl (1/10 dilution) of human feces and vortexed. Ten microliters of each spiked sample was plated in duplicate on CAT and mCCDA. The viable count of each dilution for each strain added to stool was determined by plating 10 µl of each dilution in duplicate on Columbia blood agar. Inocula were spread over the entire surface of the plates using sterile "hockey stick" spreaders and incubated at 37°C for 96 h in a microaerophilic environment.

Preliminary screening study of C. upsaliensis in humans and animals. The screening study yielded a total of six isolates of C. upsaliensis. These isolates comprised one human and five canine strains, identified from a total of 2,088 clinical specimens. The patient was a 10-year-old child with chronic diarrhea and abdominal pain. No other pathogens were identified in the stools of this child. The five dogs with C. upsaliensis in stools were asymptomatic.

Comparison of C. upsaliensis growth using the ecometric method. In the initial screening study, human clinical specimens benefited from an incubation period on CAT that was twice that of mCCDA, raising the possibility that the differences in incubation period accounted, at least in part, for the isolation of the human C. upsaliensis strain. To address this question we compared the growth of C. upsaliensis on both media after 48, 72, and 96 h of incubation using a semiquantitative technique (the ecometric method). A preliminary pilot experiment determined that growth did not occur on either medium after 24 h of incubation, and thereafter, plates were no longer examined at this time point. C. upsaliensis ATCC 43954 failed to grow on both CAT and mCCDA media AGI = 0). Growth was supported, however, on the charcoal base without cefoperazone. For the remaining 11 isolates, CAT showed a marginal benefit over mCCDA (Table 1) with 9 and 10 isolates growing well (AGI  2) at 72 and 96 h of incubation, respectively, on CAT, compared with 7 and 9 isolates with an AGI of 2 on mCCDA, after the same incubation period. Colonies at all time points tended to be smaller for C. upsaliensis growing on mCCDA than for C. upsaliensis on CAT. However, all 11 isolates were successfully cultured on both media by 96 h.

Antibiotic sensitivity testing. The six clinical isolates were subjected to antibiotic sensitivity testing using Etests. Swarming of organisms coupled with hazy growth at the edge of the zone of inhibition affected precise readings of Etest results. MICs for the human isolate were variable and showed poor reproducibility. However, all the canine isolates were resistant to cefoperazone: two strains (D4 and D6) showed no zone of inhibition on any of the medium combinations (i.e., MIC > 256 mg/liter), MICs for two isolates (D1 and D5) ranged from 64 to 256 mg/liter, and the MIC for another isolate (D2) ranged from 32 to 96 mg/liter. For both the type strain and the human clinical isolate, MICs tended to be lower when the organisms were cultured on charcoal or in the presence of teicoplanin. However, the presence of teicoplanin did not increase the MIC of cefoperazone for any strain of C. upsaliensis. The type strain, used as a control in these experiments, was cefoperazone sensitive (MIC, 0.094 to 0.75 mg/liter), thus accounting for its failure to grow on CAT medium. It was noteworthy that all clinical isolates of C. upsaliensis also were successfully subcultured on mCCDA following initial isolation on CAT.

Comparison of CAT and mCCDA for the recovery of C. upsaliensis from spiked human feces. From the fecal specimens spiked with approximately 10³ CFU of C. upsaliensis per ml all five strains tested were recovered using CAT (Table 2). However, only one of these five strains was recovered on mCCDA at this concentration. When the concentration of C. upsaliensis organisms in stools was increased to 10⁴ CFU/ml or more, recovery of all five isolates on mCCDA was achieved. Therefore, there was approximately a 1-log difference in the sensitivity of mCCDA compared to that of CAT for the recovery of C. upsaliensis from seeded stool specimens. All of the strains used in the fecal spiking experiment were resistant to cefoperazone in antibiotic disk sensitivity testing. Therefore, the increased yield of C. upsaliensis on CAT compared with mCCDA is not explained by intermediate cefoperazone sensitivities in this group of organisms.