- Email: [email protected]
Technical note: Use of a selective enrichment broth for isolation of Campylobacter species from human feces
Pathology (1985). 17, pp. 640-641
TECHNICAL NOTE: USE OF A SELECTIVE ENRICHMENT BROTH FOR ISOLATION OF CAMPYLOBACTER SPEClES FROM HUMAN FECES RACHAEL CHAN,B. HANNAN,AND R. MUNRO Bacteriology Department, Institute of Clinical Pathology and Medical Research, Westmead Centre, Sydney
Summary Isolation of Campylobacter species from 1126 fecal specimens from patients with diarrhea was compared using direct plating and selective enrichment broth. The use of the enrichment broth did not increase the isolation rate, which was 4.2%. While a selective enrichment broth may have advantages where there is delay in transit to the laboratory, or where small numbers of organisms are sought, we do not recommend its use for clinical specimens from patients with diarrhea. Key words: Campylobacter infections
Accepted May 6, 1985
INTRODUCTION Campylobacter jejuni (C. jejuni) is a well recognized cause of infectious diarrhea in Australia. In OUT laboratory it is more commonly isolated than Salmonella and Shigella species. The usual method for isolation of C. jejuni from stool specimens involves direct plating to a selective medium with incubation in a microaerophilic environment at 42 "C. A selective enrichment broth has been advocated in addition to direct plating to increase isolation of C. jejuni from human and animal specimens.'-' A selective enrichment broth is particularly useful when numbers of C. jejuni in specimens are low, or when there is delay in processing of specimens. We report an evaluation of a selective enrichment broth used in addition to direct plating for improved isolation of Campylobacter species from patients with diarrhea. Campylobacter fetus subsp. fetus was also sought, as it has recently been reported as a rare cause of infectious diarrhea, particularly in homosexual MATERIALS AND METHODS Specimens Stool specimens were received from patients with diarrhea at Westmead Hospital, Sydney, May 1984-March 1985. All specimens were examined for red cells, white cells, cysts, ova, and parasites microscopically, and cultured on selective media and in enrichment broth suitable for isolation of Salmonella species, Shigella species. Culture for Yersinia enterocolitica was performed routinely in all children less than 10 yr old, and for Aeromonas species in selected diarrheal specimens. In addition, all specimens were plated directly onto selective media and
inoculated into selective enrichment broth for isolation of Campylobacret- species. Selective media f o r Campylobacter species isolation The Campylobacter selective agar used contained Blood Agar Base No. 2 (Oxoid), 7.5% lysed horse blood with vancomycin 10 mg/l, colistin sulphate 0.3 mg/l, and trimethoprim 6.5 mg/l. The selective enrichment broth consisted of 10 ml Brucella Broth Base (Difco) with 7.5% lysed horse blood, vancomycin 10 mg/l, colistin sulphate 0.3 mg/l, and trimethoprim 6.5 mg/l. Selective plates for Campylobacter species isolation were incubated at 37 "C and 42°C in anaerobic jars evacuated and refilled with a gas mixture of 5% 0 2 , 10% CO2 and 85%N2. Plates were examined after 48 h incubation. Selective enrichment broths were incubated at 42°C in a similar microaerophilic environment for 48 h, then subcultured onto selective plates, which were incubated at 42°C microaerophilically for up to 48 h. Suspect colonies on selective plates were confirmed as C. jejuni if they showed a typical Gram stain appearance, were oxidase positive and catalase positive, were resistant to cephalothin (30 l g disc) and sensitive to nalidixic acid (30 pg disc). C. fetus subsp. fetus could be differentiated initially by its resistance to nalidixic acid and further confirmatory tests carried out.
RESULTS Eleven hundred and twenty six stool specimens were examined. The incidence of bacterial enteric pathogens is shown in Table 1. There was an overall incidence of 7.4%; Campylobacter species accounted for 4.2%, Salmonella species for 2% and Shigella species 1.O%. All Campylobacter species isolated were identified as C. jejuni. The number recovered by direct plating at 37 "C and 42°C and from enrichment broth at 42°C was identical. One isolate was recovered from enrichment TABLE1 Bacterial Enteric Pathogens Isolated from 1126 Stool Specimens Organism Campylobacter sp Salmonella sp Shigella sonnei Shigella flexneri 4 Yersinia enterocolitica Aeromonas hydrophila Total
I
Number (To) of Positive Specimens 47 22 7 5 I
(4.2%) (2.0%) (0.6%) (0.4%) (0.1%)
1 (0.1%)
83 (7.4%)
ISOLATION OF CAMPYLOBACTER SPECIES
broth only. One isolate grew only on direct plating at 37 "C and 42 "C and was not recovered from enrichment broth. Campylobacter fetus subsp. fetus was not found in any specimens. DISCUSSION We did not demonstrate any increase in recovery of C. jejuni from feces with the use of a selective enrichment broth in addition to direct plating. The recovery of bacterial enteric pathogens from hospitalized patients with diarrhea is likely t o be lower than might be expected when specimens are received from patients in the community. In our hospital practice, C. jejuni is isolated twice as frequently as Salmonella species, and accounts for over 50% of the total bacterial enteric pathogens isolated. The use of a selective enrichment broth could be easily justified if it resulted in an increased recovery of Campylobacter species. Hodge et al.7 demonstrated a 30% higher isolation rate of C. jejuni from 1249 human fecal specimens when a similar liquid enrichment broth was used, but the majority of specimens were in Cary Blair transport medium and received after an average of 4 d in transit. The advantage of the selective enrichment broth may not be so great when feces can be inoculated within a few hours of collection, as was the case with our specimens. The use of a selective enrichment broth with subculture from the broth after 24 h incubation both increases the cost of the examination and delays by up to 2 d the production of a final report. Campylobacter selective agar plates incubated at 42 "C in a microaerophilic environment are, in our experience, very inhibitory to other fecal organisms, and if Gram
641
staining is performed as an initial test on all suspect colonies, C. jejuni may be isolated even when only scanty growth occurs. We conclude that the use of a selective enrichment broth does not increase the recovery of Campylobacter species from patients with diarrhea when compared to direct plating onto selective agar. An enrichment broth may be useful if there is delay in transport of the specimen to the laboratory, or when only small numbers of the organism may be expected, as may occur with asymptomatic carriers or patients followed up after treatment. Address for correspondence: R.C., Bacteriology Department, lnstitute of Clinical Pathology and Medical Research, P.O. Box 60, Wentworthville, N.S.W., Australia 2145
References FJ, Robertson LA. Selective medium for isolating Campylobacter jejuni/coli. J Clin Pathol. 1982; 35: 462-7.
1. Bolton
2. Chan FTH, McKenzie AMR. Enrichment medium and control system for isolation of Cumpylobucferfetus subspjejuni from stools. J Clin Microbiol 1982; 15: 12-5. 3. Gilchrist MJR, Grewell CM, Washington I 1 JA. Evaluation of media for isolation of Campylobacter fetus subsp. jejuni from fecal specimens. J Clin Microbiol 1981; 14: 393-5.
4. Martin WT, Patron CM, Morris GK et al. Selective enrichment broth medium for isolation of Campylobacterjejuni. J Clin Microbiol 1983; 17: 853-5. 5. Hodge DS, Terro R. Comparative efficacy of liquid enrichment medium for isolation of Campylobacterjejuni. J Clin Microbiol 1984; 19: 434. 6. Harvey SM, Greenwood JR. Probable Cumpylobucter fetus subsp. fetus gastroenteritis. J Clin Microbiol 1983; 18: 1278-9.
I. Devlin HR, Mclntyre L. Campylobacter fetus subsp fetus in homosexual males. J Clin Microbiol 1983; 18: 999-1000.
TECHNICAL NOTE: USE OF A SELECTIVE ENRICHMENT BROTH FOR ISOLATION OF CAMPYLOBACTER SPEClES FROM HUMAN FECES RACHAEL CHAN,B. HANNAN,AND R. MUNRO Bacteriology Department, Institute of Clinical Pathology and Medical Research, Westmead Centre, Sydney
Summary Isolation of Campylobacter species from 1126 fecal specimens from patients with diarrhea was compared using direct plating and selective enrichment broth. The use of the enrichment broth did not increase the isolation rate, which was 4.2%. While a selective enrichment broth may have advantages where there is delay in transit to the laboratory, or where small numbers of organisms are sought, we do not recommend its use for clinical specimens from patients with diarrhea. Key words: Campylobacter infections
Accepted May 6, 1985
INTRODUCTION Campylobacter jejuni (C. jejuni) is a well recognized cause of infectious diarrhea in Australia. In OUT laboratory it is more commonly isolated than Salmonella and Shigella species. The usual method for isolation of C. jejuni from stool specimens involves direct plating to a selective medium with incubation in a microaerophilic environment at 42 "C. A selective enrichment broth has been advocated in addition to direct plating to increase isolation of C. jejuni from human and animal specimens.'-' A selective enrichment broth is particularly useful when numbers of C. jejuni in specimens are low, or when there is delay in processing of specimens. We report an evaluation of a selective enrichment broth used in addition to direct plating for improved isolation of Campylobacter species from patients with diarrhea. Campylobacter fetus subsp. fetus was also sought, as it has recently been reported as a rare cause of infectious diarrhea, particularly in homosexual MATERIALS AND METHODS Specimens Stool specimens were received from patients with diarrhea at Westmead Hospital, Sydney, May 1984-March 1985. All specimens were examined for red cells, white cells, cysts, ova, and parasites microscopically, and cultured on selective media and in enrichment broth suitable for isolation of Salmonella species, Shigella species. Culture for Yersinia enterocolitica was performed routinely in all children less than 10 yr old, and for Aeromonas species in selected diarrheal specimens. In addition, all specimens were plated directly onto selective media and
inoculated into selective enrichment broth for isolation of Campylobacret- species. Selective media f o r Campylobacter species isolation The Campylobacter selective agar used contained Blood Agar Base No. 2 (Oxoid), 7.5% lysed horse blood with vancomycin 10 mg/l, colistin sulphate 0.3 mg/l, and trimethoprim 6.5 mg/l. The selective enrichment broth consisted of 10 ml Brucella Broth Base (Difco) with 7.5% lysed horse blood, vancomycin 10 mg/l, colistin sulphate 0.3 mg/l, and trimethoprim 6.5 mg/l. Selective plates for Campylobacter species isolation were incubated at 37 "C and 42°C in anaerobic jars evacuated and refilled with a gas mixture of 5% 0 2 , 10% CO2 and 85%N2. Plates were examined after 48 h incubation. Selective enrichment broths were incubated at 42°C in a similar microaerophilic environment for 48 h, then subcultured onto selective plates, which were incubated at 42°C microaerophilically for up to 48 h. Suspect colonies on selective plates were confirmed as C. jejuni if they showed a typical Gram stain appearance, were oxidase positive and catalase positive, were resistant to cephalothin (30 l g disc) and sensitive to nalidixic acid (30 pg disc). C. fetus subsp. fetus could be differentiated initially by its resistance to nalidixic acid and further confirmatory tests carried out.
RESULTS Eleven hundred and twenty six stool specimens were examined. The incidence of bacterial enteric pathogens is shown in Table 1. There was an overall incidence of 7.4%; Campylobacter species accounted for 4.2%, Salmonella species for 2% and Shigella species 1.O%. All Campylobacter species isolated were identified as C. jejuni. The number recovered by direct plating at 37 "C and 42°C and from enrichment broth at 42°C was identical. One isolate was recovered from enrichment TABLE1 Bacterial Enteric Pathogens Isolated from 1126 Stool Specimens Organism Campylobacter sp Salmonella sp Shigella sonnei Shigella flexneri 4 Yersinia enterocolitica Aeromonas hydrophila Total
I
Number (To) of Positive Specimens 47 22 7 5 I
(4.2%) (2.0%) (0.6%) (0.4%) (0.1%)
1 (0.1%)
83 (7.4%)
ISOLATION OF CAMPYLOBACTER SPECIES
broth only. One isolate grew only on direct plating at 37 "C and 42 "C and was not recovered from enrichment broth. Campylobacter fetus subsp. fetus was not found in any specimens. DISCUSSION We did not demonstrate any increase in recovery of C. jejuni from feces with the use of a selective enrichment broth in addition to direct plating. The recovery of bacterial enteric pathogens from hospitalized patients with diarrhea is likely t o be lower than might be expected when specimens are received from patients in the community. In our hospital practice, C. jejuni is isolated twice as frequently as Salmonella species, and accounts for over 50% of the total bacterial enteric pathogens isolated. The use of a selective enrichment broth could be easily justified if it resulted in an increased recovery of Campylobacter species. Hodge et al.7 demonstrated a 30% higher isolation rate of C. jejuni from 1249 human fecal specimens when a similar liquid enrichment broth was used, but the majority of specimens were in Cary Blair transport medium and received after an average of 4 d in transit. The advantage of the selective enrichment broth may not be so great when feces can be inoculated within a few hours of collection, as was the case with our specimens. The use of a selective enrichment broth with subculture from the broth after 24 h incubation both increases the cost of the examination and delays by up to 2 d the production of a final report. Campylobacter selective agar plates incubated at 42 "C in a microaerophilic environment are, in our experience, very inhibitory to other fecal organisms, and if Gram
641
staining is performed as an initial test on all suspect colonies, C. jejuni may be isolated even when only scanty growth occurs. We conclude that the use of a selective enrichment broth does not increase the recovery of Campylobacter species from patients with diarrhea when compared to direct plating onto selective agar. An enrichment broth may be useful if there is delay in transport of the specimen to the laboratory, or when only small numbers of the organism may be expected, as may occur with asymptomatic carriers or patients followed up after treatment. Address for correspondence: R.C., Bacteriology Department, lnstitute of Clinical Pathology and Medical Research, P.O. Box 60, Wentworthville, N.S.W., Australia 2145
References FJ, Robertson LA. Selective medium for isolating Campylobacter jejuni/coli. J Clin Pathol. 1982; 35: 462-7.
1. Bolton
2. Chan FTH, McKenzie AMR. Enrichment medium and control system for isolation of Cumpylobucferfetus subspjejuni from stools. J Clin Microbiol 1982; 15: 12-5. 3. Gilchrist MJR, Grewell CM, Washington I 1 JA. Evaluation of media for isolation of Campylobacter fetus subsp. jejuni from fecal specimens. J Clin Microbiol 1981; 14: 393-5.
4. Martin WT, Patron CM, Morris GK et al. Selective enrichment broth medium for isolation of Campylobacterjejuni. J Clin Microbiol 1983; 17: 853-5. 5. Hodge DS, Terro R. Comparative efficacy of liquid enrichment medium for isolation of Campylobacterjejuni. J Clin Microbiol 1984; 19: 434. 6. Harvey SM, Greenwood JR. Probable Cumpylobucter fetus subsp. fetus gastroenteritis. J Clin Microbiol 1983; 18: 1278-9.
I. Devlin HR, Mclntyre L. Campylobacter fetus subsp fetus in homosexual males. J Clin Microbiol 1983; 18: 999-1000.