May 30, 1986 - Microbiol. 20:77-80. 2. Blaser, M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B.. Reller, and W. L. L. Wang. 1979. Campylobacter enteritis:.
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1987, p. 174-175 0095-1137/87/010174-02$02.00/0 Copyright C 1987, American Society for Microbiology
Vol. 25, No. 1
Evaluation of Four Enrichment Media for Isolation of Campylobacter jejuni ANDRÉS AGULLA, FRANCISCO J. MERINO, PEDRO A. VILLASANTE, JOSÉ V. SAZ, AURORA DIAZ, AURELIO C. VELASCO* Servicio de Microbiologia, Hospital de la Seguridad Social "La Paz," Madrid 28046, Spain
Received 30 May 1986/Accepted 18 September 1986
Diarrheal stool specimens were inoculated into the following media: alkaline peptone water (APW), Bruce-Zochowsky medium (BZ), Campylobacter enrichment broth (CEB), Cantpy-thio broth (CT), and Skirrow blood agar (SK) plate. All media were incubated at 42°C in microaerophilic conditions for 24 h. Afterwards, a new SK plate was inoculated from every liquid medium. Campylobacterjejuni was isolated from 43 of the 259 specimens when CT was used, from 45 when APW was used, from 46 when BZ was used, and from 46 when CEB was used; these totals include specimens that grew after enrichment only, on SK plates only, and both after enrichment and on SK plates. No significant differences were found between the isolates obtained with and without enrichment procedures.
specimens that grew after enrichment only, on SK plates only, and both after enrichment and on SK plates. No significant differences were found between the isolates obtained with and without enrichment procedures. Two isolates were recovered only in APW, three were recovered only in BZ and CEB, and none were recovered only in CT. SK plates always yielded more isolates than any enrichment medium alone. In contrast to previous reports, our results show that the use of enrichment media slightly improves the isolation rates of C. jejuni when fresh samples obtained from patients with severe gastroenteritis are used. APW and CT did not enhance the growth of C. jejuni, since many strains were lost in comparison with direct plating; BZ and CEB produced better results, but some strains were still lost. The efficacy of BZ described by Hodge and Terro (5) and that of CEB described by Martin et al. (7) greatly exceeded those in our study and could be attributed to the time taken for the transport of specimens (up to 21 days in Cary-Blair transport medium). In our study, stool specimens were always inoculated within a few hours after collection. Rubin and Woodard (9) reported that refrigeration of CT at 4°C increased the isolation rate of C. jejuni. However, similar to our experience, Luechtefeld et al. (6) found that the results obtained after incubating CT at 42°C in a microaerophilic atmosphere were disappointing. APW has been used as an enrichment medium for Vibrio cholerae, but it is not efficient for the recovery of thermophilic campylobacters, as previously reported by Chan and Mackenzie (4). Although BZ and CEB produced better results, we agree with Bolton and Robertson (3), who felt that enrichment media were not useful for the diagnosis of human infections. In our opinion, enrichment media may be useful just for samples that contain very few viable organisms, specimens which have been delayed in transport, or feces of patients previously treated. They may also be valuable in epidemiological studies. On the other hand, when fresh samples from patients with acute diarrhea are received, the use of enrichment media is not advisable, owing to the low number of additional isolations as compared with direct plating.
solid media is the standard technique used in
most clinical laboratories for the isolation of Campylobacter jejuni from human stools. Previous reports (1, 4, 5, 7, 9)
showed that specific enrichment media increase the number of Campylobacter isolates recovered. In the present work, we evaluate the efficiency of four enrichment media and compare them with direct inoculation on a widely used solid medium. A total of 259 stool specimens were selected from untreated patients suffering from acute diarrhea and having no underlying disease. Within a maximum of 4 h after collection, a heavy suspension of the samples was made in saline solution. Three to four drops of this suspension was inoculated into 10 ml of the following media: alkaline peptone water (APW) (8), Bruce-Zochowsky medium (BZ) (5), which contains nutrient broth 2 (Oxoid Ltd., Basingstoke, England) supplemented with 7% horse blood and 10 mg of vancomycin, 2,500 IU of polymyxin, and 25 mg of trimethoprim per liter; Campylobacter enrichment broth (CEB) (7), which contains brucella broth (Difco Laboratories, Detroit, Mich.) and 333 mg of 5-fluorouracil, 32 mg of cefoperazone, and 32 mg of trimethoprim per liter; and Campy-thio (CT) (2), which contains 10 mg of vancomycin, 2,500 IU of polymyxin, 5 mg of trimethoprim, 2 mg of amphotericin B, and 15 mg of cephalothin per liter. One drop was used to inoculate a Skirrow blood agar (SK) plate (10). All media were incubated at 42°C in microaerophilic conditions (5% Or-10% C02) provided by a gas generating kit and an anaerobic catalyst (Oxoid) for 24 h. Afterwards, a new SK plate was inoculated from every liquid medium. Plates showing no growth were incubated for up to 48 h. A Campylobacter strain was inoculated daily into al media used for quality control. The maximum storage time of plates and liquid media was 5 days. The results are shown in Table 1. A total of 45 specimens were positive when APW was used, 46 were positive when BZ was used, 43 were positive when CT was used, and 46 were positive when CEB was used; these totals include *
Corresponding author. 174
VOL. 25, 1987 TABLE 1. Isolation of C. jejuni in four enrichment media (n = 259) Medium
After enrichment only
APW BZ CT CEB
2 3 0 3
No. of isolates that grew: After enrichment On SK plate and on SK plate only
29 39 10 36
14 4 33 7
This work was supported in part by Ahorros y Monte de Piedad de Madrid.
grant from the Caja de
LITERATURE CITED 1. Barot, M. S., and V. D. Bokkenheuser. 1984. Systematic investigation of enrichment media for wild-type Campylobacterjejuni strains. J. Clin. Microbiol. 20:77-80. 2. Blaser, M. J., I. D. Berkowitz, F. M. LaForce, J. Cravens, L. B. Reller, and W. L. L. Wang. 1979. Campylobacter enteritis: clinical and epidemiological features. Ann. Intern. Med. 91:179-185.
3. Bolton, F. J., and L. Robertson. 1982. A selective medium for isolating Campylobacterjejunilcoli. J. Clin. Pathol. 35:462-467. 4. Chan, F. T. H., and A. M. R. Mackenzie. 1982. Enrichment medium and control system for isolation of Campylobacterfetus subsp. jejuni from stools. J. Clin. Microbiol. 15:12-15. 5. Hodge, D. S., and R. Terro. 1984. Comparative efficacy of enrichment medium for isolation of Campylobacter jejuni. J. Clin. Microbiol. 19:434. 6. 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. 7. 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. 8. Phillips, E., and P. Nash. 1985. Culture media, p. 1051-1092. In E. H. Lennette, A. Balows, W. J. Hausler, and H. J. Shadomy (ed.), Manual of clinical microbiology, 4th ed. American Society for Microbiology, Washington, D.C. 9. Rubin, S. J., and M. Woodard. 1983. Enhanced isolation of Campylobacterjejuni by cold enrichment in Campy-thio broth. J. Clin. Microbiol. 18:1008-1010. 10. Skirrow, M. B. 1977. Campylobacter enteritis: a "new" disease. Br. Med. J. 2:9-11.