of Campylobacterjejuni from Dogs - Journal of Clinical Microbiology

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Two techniques for the isolation of Campylobacterjejuni from feces, direct plating and thioglycolate broth enrichment, were compared. A total of 272 rectal swab ...
Vol. 26, No. 11

JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1988, p. 2246-2247

0095-1137/88/112246-02$02.00/0 Copyright © 1988, American Society for Microbiology

Comparison of Broth Enrichment and Direct Plating for the Isolation of Campylobacter jejuni from Dogs J. DANIEL MONFORT,* HAROLD F. STILLS, JR., AND STEEN BECH-NIELSEN Department of Veterinary Preventive Medicine, College of Veterinary Medicine, Ohio State University, 1900 Coffey Road, Columbus, Ohio 43210 Received 13 June 1988/Accepted 8 August 1988

Two techniques for the isolation of Campylobacterjejuni from feces, direct plating and thioglycolate broth enrichment, were compared. A total of 272 rectal swab cultures were performed on 156 laboratory dogs. Campylobacter blood agar plates and Campylobacter thioglycolate broth were inoculated immediately upon sampling of the dogs. After incubation at 40C for 12 to 16 h, material from the Campylobacter thioglycolate medium was inoculated onto Campylobacter blood agar plates. A total of 157 samples were positive for C. jejuni; 154 were positive by the direct method and 112 were positive by the enrichment technique. Forty-five samples which were negative by the enrichment method were positive by the direct method, and three samples which were negative by the direct method were positive for C. jejuni by the enrichment method. The use of the enrichment step resulted in an increase in the isolation rate from 56.6 to 57.7%.

provided by thrice evacuating anaerobic jars (BBL GasPak system) to a pressure of 250 mm Hg (33,320 Pa) and filling with an analyzed mixture of 5.07% oxygen, 10.21% carbon dioxide, and 84.72% nitrogen. Inoculated CTB tubes were incubated at 4°C for 12 to 16 h, and approximately 0.5 ml of each was used to inoculate Campy-BAP. All plates were incubated for 72 h and observed daily for growth. Positive controls of C. jejuni ATCC 29428 were included in each container of plates. Organisms were identified as C. jejuni by the following criteria: growth at 42°C, typical colonial and cellular morphology, oxidase positivity, catalase positivity, production of hydrogen sulfide gas (as detected on lead acetate paper), and the ability to hydrolyze sodium hippurate (3).

Enrichment media are commonly used in human and veterinary clinical laboratories to increase the isolation rates for Campylobacterjejuni. The expense and effort involved in the use of an enrichment step require that its benefit be demonstrated. Previously, investigators have reported various degrees of improvement over direct plating by the use of several enrichment media, including Campylobacter thioglycolate broth (CTB) (5, 11), Campylobacter enrichment broth (9), Bruce-Zochowsky medium (6), alkaline peptone water (7), and others (4, 8-10), when attempting to isolate C. jejuni from human stool specimens. However, Agulla et al. (1) reported no significant difference in the Campylobacter isolation rates from human stool when using an enrichment procedure, and Leuchtefeld et al. (8) reported a similar lack of improvement over direct plating when sampling turkeys. In this work we evaluate the effectiveness of CTB and compare it with direct inoculation on Campylobacter blood agar plates (Campy-BAP) plates when used for the isolation of C. jejuni from a colony of laboratory dogs endemically infected with C. jejuni.

RESULTS Of the 272 samples processed, 157 (57.7%) were positive for C. jejuni by either method. A total of 154 (56.6%) were positive by the direct plating method, and 112 (41.2%) were positive by the CTB enrichment technique. One hundred nine samples (40.1%) were positive by both direct and enrichment techniques. Forty-five samples (16.5%) which were negative via the enrichment technique were positive by the direct method. Three samples (1.1%) which were negative by direct plating were positive by the enrichment

MATERIALS AND METHODS Animals. A total of 272 stool specimens were acquired with rectal swabs from 156 diarrheic dogs in a laboratory animal setting over a 4-month period. C. jejuni had previously been isolated from several dogs in the colony. Although the dogs sampled were passing poorly formed stool, they were otherwise clinically normal. None were anorectic or lost weight during the period of this study. Media. Campy-BAP were prepared with brucella agar base (CM169; Oxoid U.S.A., Columbia, Md.), 10% sheep blood, and Blaser-Wang Campylobacter selective supplement (SR98; Oxoid U.S.A.). Two separate rectal swabs were taken from each dog. The first swab was used for the direct inoculation of Campy-BAP. The second swab was used for the inoculation of CTB (21748; BBL Microbiology Systems, Cockeysville, Md.). All broth and solid media were placed in an ice chest for a period riot exceeding 1 h during transportation to the laboratory. Inoculated Campy-BAP were incubated at 42°C in a microaerobic environment *

technique. DISCUSSION Although earlier studies found a significant improvement in isolation rates when enrichment techniques were used (4, 6-11), recent reports have shown no such benefit in a clinical setting. Bolton and Robertson (2) concluded that when culturing diarrheic individuals, no benefit was derived from the use of an enrichment step. When culturing human fecal samples, Agulla et al. (1) reported no isolation rate increase over direct plating when CTB at 42°C was used. The recommended (by the. manufacturer) incubation temperature of 4°C employed in this study also did not markedly increase the isolation rate. Only 3 of the 157 positive cultures resulted from the enrichment step, an increase of 1.9% over direct plating alone.

Corresponding author. 2246

VOL . 26 1988 ,

CAMPYLOBACTER JEJUNI: ENRICHMENT VERSUS DIRECT PLATING

We believe that the apparent discrepancies between this study and those which reported an advantage to enrichment are due to differences in culture technique. Immediate plating of rectal swabs has been shown to yield higher isolation rates than the culture of fecal specimens (7). In this study, rectal swabs were immediately plated following collection and placed in a microaerobic environment within 1 h, thereby reducing the exposure of the organisms to atmospheric levels of oxygen. The evacuation and replacement technique employed supplied the proper atmosphere in less than 2 min; atmosphere generators require considerably more time. For example, the manufacturer of the CampyGasPak (71034; BBL) states that the proper atmosphere will be generated in less than 1 h. Direct plating also eliminates the need for transport medium, which may cause a reduction in the rate of recovery of C. jejuni. The use of separate swabs for the inoculation of the two types of media prevented simultaneous exposure of the organisms to the antimicrobial effects of both media. Also, all samples were refrigerated until a microaerobic atmosphere and incubation temperature were provided. These procedures all serve to reduce the damage to Campylobacter organisms, increasing culture positivity of direct plating and thereby diminishing the impact of enrichment, which may serve to restore damaged cells to a more vigorous condition. This study supports the conclusions of Agulla et al. and Bolton and Robertson in that we could not demonstrate any value in the use of CTB for the isolation of C. jejuni from diarrheic dogs. In light of the expense and the time involved in enrichment procedures, we subscribe to their use only when an unavoidable delay between specimen collection and plating on selective medium takes place, which reduces the number of viable Campylobacter organisms. In addition to the lack of evidence supporting the use of enrichment procedures for the isolation of C. jejuni from diarrheic stool, it is obvious from previous studies (1, 6, 8, 9, 11) and from this study that direct plating of fecal specimens is necessary whether or not enrichment is used. If direct plating were not performed in this study, the number of C. jejuni isolations would have been 112 of 272 instead of 157 of

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272, a 28.7% reduction in the number of positive cultures (45 of 157; P < 0.05, McNemar's test). ACKNOWLEDGMENT This work was supported by a grant from the Canine Research Fund of the Ohio State University College of Veterinary Medicine. LITERATURE CITED 1. Agulla, A., F. J. Merino, P. A. Villasante, J. V. Saz, A. Diaz, and A. C. Velasco. 1987. Evaluation of four enrichment media for isolation of Campylobacter jejuni. J. Clin. Microbiol. 25:174175. 2. Bolton, F. J., and L. Robertson. 1982. A selective medium for isolating Campylobacterjejuni/coli. J. Clin. Pathol. 35:462-467. 3. Butzler, J.-P. (ed.). 1984. Campylobacter infections in man and animals, p. 46-47. CRC Press, Inc., Boca Raton, Fla. 4. Chan, F. T. H., and A. M. R. MacKensie. 1982. Enrichment medium and control system for isolation of Campylobacterfetus subsp. jejuni from stools. J. Clin. Microbiol. 15:12-15. 5. Gilchrist, M. J. R., C. M. Grewell, and J. A. Washington IL. 1981. Evaluation of media for isolation of Campylobacterfetus subsp. jejuni from fecal specimens. J. Clin. Microbiol. 14:393395. 6. Hodge, D. S., and R. Terro. 1984. Comparative efficacy of liquid enrichment medium for isolation of Campylobacter jejuni. J. Clin. Microbiol. 19:434. 7. Kaplan, R. L., L. J. Goodman, J. E. Barrett, G. M. Trenholme, and W. Landau. 1982. Comparison of rectal swabs and stool cultures in detecting Campylobacterfetus subsp. jejuni. J. Clin. Microbiol. 15:959-960. 8. Luechtefeld, N. W., W. L. L. Wang, M. J. Blaser, and L. B. Reller. 1981. Evaluation of transport and storage techniques for isolation of Campylobacterfetus subsp. jejuni from turkey fecal specimens. J. Clin. Microbiol. 13:438 443. 9. Martin, W. T., C. M. Patton, G. K. Morris, M. E. Potter, and N. D. Puhr. 1983. Selective enrichment broth medium for isolation of Campylobacterjejuni. J. Clin. Microbiol. 17:853-855. 10. Rogol, M., B. Shpak, D. Rothman, and I. Sechter. 1985. Enrichment medium for isolation of Campylobacter jejuni-Campylobacter coli. Apple. Environ. Microbiol. 50:125-126. 11. Rubin, S. J., and M. Woodard. 1983. Enhanced isolation of Campylobacterjejuni by cold enrichment in Campy-thio broth. J. Clin. Microbiol. 18:1008-1010.