SOP: Isolation of Arcobacter Species from Foods

GIFSQ — Global Institute for Food Safety & Quality

1. Principle

This SOP isolates Arcobacter species from foods following the APHA Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 30, 4th Edition. Two enrichment-based procedures are given. The first uses a two-step approach: selective enrichment in Ellinghausen-McCullough-Johnson-Harris polysorbate 80 (EMJH-P80) semi-solid medium, examined microscopically or by PCR, followed by streaking to cefoperazone-vancomycin-amphotericin B (CVA) agar or modified charcoal-cefoperazone-deoxycholate (mCCDA) agar. The second, the Johnson and Murano (JM) protocol, enriches in JM semi-solid broth and plates to charcoal-based JM agar, with plating in air to quench oxygen. Colonies are confirmed by phenotypic tests (indoxyl acetate hydrolysis, oxidase, catalase, cadmium chloride sensitivity) and PCR. Arcobacter species are distinguished from Campylobacter by their ability to grow in air at 25 °C to 37 °C.

2. Apparatus required

• Sterile pipettes and Pasteur pipettes
• Sterile Petri dishes and culture tubes
• Stomacher laboratory blender and sterile stomacher bags
• Incubator capable of maintaining 25 °C to 30 °C
• Microaerobic incubation jars or pouches (CampyGas Pak)
• Darkfield microscope, 400× magnification
• 0.45 µm membrane filters (Millipore HAWP)

3. Reagents

• A. EMJH-P80 semi-solid medium — Solution A: sterile distilled water — 700 mL; 25× phosphate buffered solution — 40 mL; 20× salt solution — 50 mL; copper solution — 1 mL; zinc solution — 10 mL; iron solution — 20 mL; L-cystine — 0.2 g. Solution B: sterile distilled water — 120 mL; B-12 vitamin solution — 20 mL; thiamine HCl B-1 solution — 0.1 mL; Tween 80 — 1.2 mL
• To prepare: autoclave; stir Solution A for 5 min until all the cystine dissolves; filter Solution A through triple-thickness Whatman No. 1 filter paper until clear; add Solution A to Solution B and add distilled water to bring to a final volume of 1 L; dispense 800 mL of this final product into a 2 L Erlenmeyer flask; add 1.3 g of agar and adjust the pH to 7.2–7.4; autoclave at 121 °C for 15 min; cool to 50 °C in a water bath; add 200 mg of 5-fluorouracil and filter-sterilized 5% albumin solution; dispense into sterile tubes, 10 mL per tube
• B. CVA (cefoperazone vancomycin amphotericin B blood) agar — base: tryptone — 10.0 g; peptamin — 10.0 g; dextrose — 1.0 g; yeast extract — 2.0 g; sodium chloride — 5.0 g; sodium bisulfite — 0.1 g; agar — 15.0 g; distilled water — 1.0 L
• Antibiotic stock solution: cefoperazone — 2.0 g; vancomycin — 1.0 g; amphotericin B — 0.2 g; bring to 100 mL with sterile water; filter-sterilize and dispense in 3.0 mL aliquots; stable at −20 °C for 3 months
• To prepare: prepare the base agar in distilled water, autoclave (121 °C for 20 min) and cool at 47 °C; add 5% defibrinated bovine or ovine blood; add 1.0 mL of the CVA antibiotic stock solution to 1 L of agar to give final concentrations of cefoperazone 20 mg/L, vancomycin 10 mg/L, and amphotericin B 2 mg/L; dispense to Petri plates (100 mm × 15 mm); also available commercially (Remel 01-270)
• C. mCCDA agar, commercially available (Remel), for plating from EMJH-P80
• D. Brain heart infusion (BHI) agar with 0.6% yeast extract and 10% defibrinated bovine or ovine blood, for the membrane filtration procedure
• E. BHI agar supplemented with 5% defibrinated bovine blood, for the cadmium chloride test
• F. JM semi-solid enrichment broth: special peptone — 10.0 g; yeast extract — 5.0 g; beef extract — 5.0 g; sodium chloride — 4.0 g; potassium phosphate monobasic — 1.5 g; sodium phosphate dibasic — 3.5 g; pyruvate — 0.5 g; thioglycollate — 0.5 g; charcoal — 0.5 g; bile salts No. 3 — 2.0 g; agar — 2.0 g; distilled water — 1.0 L; with antimicrobials at final concentrations of 5-fluorouracil — 200 mg/L and cefoperazone — 32 mg/L
• To prepare: mix the ingredients (except the antimicrobials) with distilled water to a final volume of 1.0 L (pH 6.8); heat to boiling and cool to 55–60 °C; stir the mixture to keep the charcoal in suspension, since the charcoal will not completely dissolve; autoclave at 121 °C for 20 min and cool to 55–60 °C; stir and add the antimicrobials to the desired concentrations. For the cefoperazone stock, dissolve 320 mg of cefoperazone in 10 mL of distilled water, filter-sterilize through a 0.45 µm filter, and store in screw-cap test tubes (20 mm × 125 mm)
• G. JM agar (plating medium): special peptone — 10.0 g; yeast extract — 5.0 g; beef extract — 5.0 g; sodium chloride — 4.0 g; potassium phosphate monobasic — 1.5 g; sodium phosphate dibasic — 3.5 g; pyruvate — 0.5 g; thioglycollate — 0.5 g; agar — 20.0 g; distilled water — 1.0 L (no preparation procedure is given in the media chapter)
• H. Arcobacter Selective Media, commercially available (Oxoid CM0965B): peptone base with cefoperazone, amphotericin B, and teicoplanin; contains no oxygen-quenching system
• I. Buffered peptone water (BPW), for the 1:2 sample dilution in the JM procedure
• J. Cary-Blair transport medium, commercial, for transport of cultures
• K. Heart infusion agar or BHI slants, for the catalase test
• L. MacConkey agar, for the identification tests
• M. Campylobacter minimal medium, for the identification tests
• N. Cadmium chloride test disks: add cadmium chloride to filter paper disks to a final concentration of 2.5 µg and allow to dry
• O. Indoxyl acetate hydrolysis test reagents
• P. 3% hydrogen peroxide solution, for the catalase test
• Q. Oxidase test reagent

4. Sample preparation

For the EMJH-P80 procedure, place livestock samples, faeces, cloacal swabs, or tissue samples (about 1 g) in 9 mL of EMJH-P80 medium. For the JM procedure, prepare a 1:2 dilution of the sample in buffered peptone water and stomach the mixture. Use Cary-Blair transport medium for transport of cultures.

5. Procedure

5.1. EMJH-P80 two-step procedure

Incubate the EMJH-P80 tube aerobically at 30 °C for 3 to 5 days
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Examine by darkfield microscopy or by PCR: prepare wet mounts and examine under 400× darkfield for slender, curved rods with rapid darting motility; helical chains moving with a spiral motion may also be present (motility is reduced when the microbes are trapped and concentrated in the agar)
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For bacterial isolation, streak an aliquot from the EMJH-P80 onto the surface of CVA agar or mCCDA agar
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Incubate microaerobically in a CampyGas Pak at 25–30 °C for 2 days
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If the sample is heavily contaminated, isolate Arcobacter from the EMJH-P80 by placing a 0.45 µm membrane filter onto the surface of BHI agar supplemented with 0.6% yeast extract and 10% defibrinated bovine or ovine blood: with a sterile cotton-plugged Pasteur pipette, remove an aliquot from the zone of growth below the surface of the EMJH-P80, place a drop or two of the culture fluid on the filter, and leave the filter undisturbed for 1 hour at room temperature so that Arcobacter swim through the membrane filter
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Remove the filter and streak for isolation, using the area where the filter was placed as the first quadrant; incubate at 30 °C for 2 days microaerobically
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Examine for colonies 1 to 2 mm in diameter, ranging in colour from white translucent to buff; colonies may appear to swarm

5.2. Johnson and Murano (JM) procedure

Place a 1 mL aliquot of the stomached 1:2 sample dilution into 9 mL of JM semi-solid enrichment medium
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Incubate aerobically for 48 hours at 30 °C
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Remove a loopful of culture from 1 cm below the surface and streak onto JM plating medium
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Incubate in air for 48 hours at 30 °C
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Pick presumptive colonies — grey or off-white, round, with or without a surrounding red colour — and purify
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Test the colonies for oxidase and catalase activity and prepare a Gram stain
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Confirm identification by the multiplex PCR assay for Arcobacter and Arcobacter butzleri, including strains of A. butzleri ATCC 49616, A. cryaerophilus 1A ATCC 43158, and A. cryaerophilus 1B ATCC 43157 as reference standards

5.3. Commercial Arcobacter selective medium variant

Plate the sample on Arcobacter Selective Media (Oxoid CM0965B): the peptones in the base medium facilitate the growth of Arcobacter species while the cefoperazone, amphotericin B, and teicoplanin inhibit competing flora
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Incubate in air at 30 °C for 24 hours (the absence of an oxygen-quenching system suppresses the growth of Campylobacter)

5.4. Identification of suspect colonies

Perform indoxyl acetate hydrolysis: Arcobacter species hydrolyse indoxyl acetate, shown by a blue colour
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Perform the oxidase test: all Arcobacter species are oxidase-positive
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Perform the catalase test by adding several drops of 3% hydrogen peroxide to 24–48 hour growth on a heart infusion agar (BHI) slant; production of any bubbles is considered positive — A. cryaerophilus and A. skirrowii are catalase-positive, while A. butzleri is weak or negative for catalase production
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Perform the cadmium chloride test: place a disk carrying 2.5 µg of cadmium chloride on BHI agar supplemented with 5% defibrinated bovine blood that has been inoculated with the test organism, and incubate at 30 °C for 1 to 2 days — A. cryaerophilus is sensitive to cadmium chloride, A. butzleri is resistant
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The most reliable tests to identify A. butzleri are negative or weak catalase production, growth in Campylobacter minimal medium, abundant growth on blood agar, growth on MacConkey agar, and resistance to cadmium chloride; in contrast, A. cryaerophilus is strongly catalase-positive and does not grow on Campylobacter minimal medium or on MacConkey agar
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Species of Arcobacter are differentiated from Campylobacter by their ability to grow in air at 25 °C to 37 °C

6. Precautions

• Scan darkfield fields carefully for agar particles, which can be mistaken for organisms.
• The JM protocol was more sensitive than EMJH-P80 in detecting Arcobacter from heavily contaminated poultry.
• For PCR confirmation, a 1223 bp amplicon is specific for the genus Arcobacter; A. butzleri additionally shows a 686 bp product unique to the species.

7. References

• APHA, Compendium of Methods for the Microbiological Examination of Foods (CMMEF), Chapter 30, 4th Edition.