Specimen Collection, Transport, and Processing
There are no special requirements for the collection, transport, and processing of clinical specimens for the detection of campylobacters; the two most common clinical specimens submitted to the laboratory are feces (rectal swabs are also acceptable for culture) and blood. Specimens should be processed as soon as possible. Delays of more than 2 hours require the stool specimen to be placed either in Cary-Blair transport medium or in campy thio, a thioglycollate broth base with 0.16% agar and vancomycin (10 mg/L), trimethoprim (5 mg/L), cephalothin (15 mg/L), polymyxin B (2500 U/L), and amphotericin B (2 mg/L). Cary-Blair transport medium is suitable for other enteric pathogens; specimens received in transport medium should be processed immediately or stored at 4° C until processed.
Direct Detection
Upon gram staining, Campylobacter spp. display a characteristic microscopic morphology as small, curved or seagull-winged, faintly staining, gram-negative rods (Figure 1). Polymerase chain reaction (PCR) amplification may provide an alternative to culture methods for the detection of Campylobacter spp. from clinical specimens. The detection of Campylobacter DNA in stools from a large number of patients with diarrhea suggests that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiologic agent is identified.
Fig1. A, Gram stain appearance of Campylobacter jejuni subsp. jejuni from a colony on a primary isolation plate. Note seagull and curved forms (arrows). B, Appearance of Campylobacter jejuni subsp. jejuni in a direct Gram stain of stool obtained from a patient with campylobacteriosis. Arrows point to the seagull form.
Antigen Detection
Several commercial antigen detection systems are available for the direct detection of Campylobacter in stool specimens. These enzyme immunoassays (EIA) can be used to detect antigens in stool samples for several days if stored at 4° C.
Media
Campy-BAP is an enriched selective blood agar plate used to isolate C. jejuni. The medium is composed of a Brucella agar base, sheep red blood cells and vancomycin, trimethoprim, polymyxin B, amphotericin B, and cephalothin. Campy medium (CVA) contains cefoperazone, vancomycin, and amphotericin B. The antibiotics in both media suppress the growth of normal fecal flora. Campylobacter agar base blood free (CCDA) is a modified agar that does not include blood. The blood is replaced with charcoal, sodium pyruvate, and ferrous sulfate. The medium supports growth of most Campylobacter spp.
Cultivation
Stool. Successful isolation of Campylobacter spp. from stool requires selective media and optimum incubation conditions. Recommended inoculation of two selective agars is associated with increased recovery of the organisms. Because Campylobacter and Arcobacter spp. have different optimum temperatures, two sets of selective plates should be incubated, one at 42° C and one at 37° C. Extended incubation may be required, 48 to 72 hours, before there is evidence of visible growth. Table 1 describes the selective plating media and incubation conditions required for the recovery of Campylobacter spp. from stool specimens.
Table1. Selective Media and Incubation Conditions to Recover Campylobacter and Arcobacter spp. from Stool Specimens
A filtration method can also be used in conjunction with a nonselective medium to enhance recovery of Campylobacter and Arcobacter spp. A filter (0.65-μm pore-size cellulose acetate) is placed on the agar surface, and a drop of stool is placed on the filter. The plate is incubated upright. After 60 minutes at 37° C, the filter is removed and the plates are reincubated in a microaerobic atmosphere. The organisms are motile and capable of migrating through the filter, producing isolated colonies on the agar surface and effectively removing contaminating stool flora. C. concisus, A. butzleri, A. cryaerophilus, and H. cinaedi have been isolated following 5 to 6 days of incubation using the filter technique. An enrichment broth may also be used for the recovery of Arcobacter or Campylobacter species from stool.
Blood. Campylobacter spp. are capable of growth in less than 5 days in most blood culture media, although they may require extended incubation periods of up to 2 weeks for detection. Subcultures should be incubated in 5% to 10% O2 (microaerobic) environment. Turbidity may not visible in blood culture media; therefore, blind subcultures or microscopic examination using acridine orange stain may be necessary. The presence of Campylobacter spp. in blood cultures is effectively detected through carbon dioxide (CO2) monitoring. Isolation from sources other than blood or feces is extremely rare. Recovery of the organisms is enhanced by inoculation (minced tissue, wound exudate) to a nonselective blood or chocolate agar plate and incubation at 37° C in a CO2-enriched, microaerobic atmosphere. (Selective agars containing a cephalosporin, rifampin, and polymyxin B may inhibit growth of some strains and should not be used for isolation from sterile sites.)
Approach to Identification
Plates should be examined for characteristic colonies, which are gray to pinkish or yellowish gray and slightly mucoid looking; some colonies may exhibit a tailing effect along the streak line (Figure 2). Colony morphology varies with the type of medium used for isolation. Suspicious-looking colonies observed on selective media incubated at 42° C may be presumptively identified as Campylobacter spp., usually C. jejuni or C. coli, with a few basic tests. A wet preparation of the organism in broth may be examined for characteristic darting motility and curved morphology on Gram stain. Both organ isms are cephalothin resistant, nalidixic acid sensitive, and sensitive to lysis by complement. C. fetus is incapable of growth at 42°, and optimal growth is 37°; it is cephalothin sensitive, nalidixic acid resistant, and resistant to complement lysis.
Fig2. Colonies of Campylobacter jejuni following 48 hours of incubation on a selective medium in a microaerobic atmosphere.
Almost all the pathogenic Campylobacter spp. are oxidase positive and catalase positive. Frequently laboratories will report stool isolates as “Campylobacter spp.”
Most Campylobacter spp. are asaccharolytic, unable to grow in 3.5% NaCl, although strains of Arcobacter appear more resistant to salt and, except for Arcobacter cryaerophilus, unable to grow in ambient air. Growth in 1% glycine is variable. Susceptibility to nalidixic acid and cephalothin, as previously described (Table2), is determined by inoculating a 5% sheep blood or Mueller-Hinton agar plate with a McFarland 0.5 turbidity suspension of the organism, placing 30-mg disks on the agar surface and incubating in 5% to 10% CO2 at 37° C. Other tests useful for identifying these species are the rapid Hippurate hydrolysis test, production of hydrogen sulfide (H2S) in triple sugar iron agar slants, nitrate reduction, and hydro lysis of indoxyl acetate. Indoxyl acetate disks are available commercially. Cellular fatty acid analysis is useful for species identification. This method is not available in routine clinical microbiology laboratories. Several commercial products are available for species identification, including particle agglutination methods and nucleic acid probes.
Table2. Differential Characteristics of Clinically Relevant Campylobacter, Arcobacter, and Helicobacter spp.
Molecular assays based on PCR amplification of the 16S rRNA gene and direct sequencing of the PCR product have successfully been used to identify the majority of Campylobacter species. The assays accurately discriminate related taxa including Campylobacter, Arcobacter, or Helicobacter species. Finally, another approach using 16S-23S PCR-based amplification with a DNA probe colorimetric membrane assay proved to rapidly detect and identify Campylobacter in stool specimens.
Serodiagnosis
Serodiagnosis is not widely applicable for the diagnosis of infections caused by these organisms.
