International Journal of Food Microbiology 189 (2014) 67–74

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Development of a selective agar plate for the detection of Campylobacter spp. in fresh produce Jin-Hee Yoo, Na-Young Choi, Young-Min Bae, Jung-Su Lee, Sun-Young Lee ⁎ Department of Food Science and Technology, Chung-Ang University, 72-1 Nae-ri, Daedeok-myeon, Anseong-si, Gyeonggi-do 456-756, South Korea

a r t i c l e

i n f o

Article history: Received 31 March 2014 Received in revised form 17 July 2014 Accepted 24 July 2014 Available online 2 August 2014 Keywords: Campylobacter Selective medium Bolton agar Antibiotic

a b s t r a c t This study was conducted to develop a selective medium for the detection of Campylobacter spp. in fresh produce. Campylobacter spp. (n = 4), non-Campylobacter (showing positive results on Campylobacter selective agar) strains (n = 49) isolated from fresh produce, indicator bacteria (n = 13), and spoilage bacteria isolated from fresh produce (n = 15) were plated on four Campylobacter selective media. Bolton agar and modified charcoal cefoperazone deoxycholate agar (mCCDA) exhibited higher sensitivity for Campylobacter spp. than did Preston agar and Hunt agar, although certain non-Campylobacter strains isolated from fresh produce by using a selective agar isolation method, were still able to grow on Bolton agar and mCCDA. To inhibit the growth of non-Campylobacter strains, Bolton agar and mCCDA were supplemented with 5 antibiotics (rifampicin, polymyxin B, sodium metabisulfite, sodium pyruvate, ferrous sulfate) and the growth of Campylobacter spp. (n = 7) and non-Campylobacter strains (n = 44) was evaluated. Although Bolton agar supplemented with rifampicin (BR agar) exhibited a higher selectivity for Campylobacter spp. than did mCCDA supplemented with antibiotics, certain non-Campylobacter strains were still able to grow on BR agar (18.8%). When BR agar with various concentrations of sulfamethoxazole–trimethoprim were tested with Campylobacter spp. (n = 8) and non-Campylobacter (n = 7), sulfamethoxazole–trimethoprim was inhibitory against 3 of 7 non-Campylobacter strains. Finally, we validated the use of BR agar containing 50 mg/L sulfamethoxazole (BRS agar) or 0.5 mg/L ciprofloxacin (BRCS agar) and other selective agars for the detection of Campylobacter spp. in chicken and fresh produce. All chicken samples were positive for Campylobacter spp. when tested on mCCDA, BR agar, and BRS agar. In fresh produce samples, BRS agar exhibited the highest selectivity for Campylobacter spp., demonstrating its suitability for the detection of Campylobacter spp. in fresh produce. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Campylobacter is a Gram negative, non-spore forming, oxidasepositive, and microaerophilic bacterium. The Campylobacter is 0.2– 0.8 μm in width and 0.5–5 μm in length, with a spiral, curved, or rodshaped appearance (Keener et al., 2004; Ryan and Ray, 2004). Pathogenic infection by Campylobacter spp. can result in campylobacteriosis, a gastrointestinal disease characterized by profuse, and often bloody diarrhea, and particularly in children, clinical symptoms of acute abdominal pain and fever are observed after 4 days (Adak et al., 2002). The species most often implicated as the causative agent of campylobacteriosis is Campylobacter jejuni, followed by Campylobacter coli, and relatively infrequently, Campylobacter upsaliensis and Campylobacter lari (Labarca et al., 2002; Prasad et al., 2001; Vandamme, 2000). The rate of campylobacteriosis has been increasing worldwide, with the number of cases often exceeding those of salmonellosis and shigellosis (Cover et al., 2014). In the United States, campylobacteriosis is the third most frequent bacterial foodborne disease, with 800,000 ⁎ Corresponding author. Tel.: +82 31 670 4587; fax: +82 31 676 8741. E-mail addresses: [email protected], [email protected] (S.-Y. Lee).

http://dx.doi.org/10.1016/j.ijfoodmicro.2014.07.032 0168-1605/© 2014 Elsevier B.V. All rights reserved.

estimated cases per year, accounting for 8% of the overall estimated foodborne diseases (Scallan et al., 2011). In rare cases, campylobacteriosis is associated with severe disabling consequences such as septicemia, irritable bowel syndrome, reactive arthritis, or autoimmune neuropathies (e.g., Guillain-Barré syndrome and Reiter's syndrome) (Humphrey et al., 2007; Takahashi et al., 2005). The major risk factors for campylobacterios in humans are consumption of undercooked poultry, untreated or contaminated water, and raw or improperly pasteurized milk (Butzler, 2004; Friedman et al., 2004). In recent years, the demand for ready-to-eat fresh produce has risen. Organic agriculture has also increased in many countries. Given that poultry manure is often used for the cultivation of fresh produce, consumption of fresh produce is likely to lead to Campylobacter exposure. Accordingly, the development of accurate detection methods for effective monitoring and risk assessment of Campylobacter contamination in fresh produce is needed. However, the isolation of Campylobacter spp. in food is complicated because of the low number of these bacteria in food such as poultry, meat, and milk; the high number of competitor organisms; and the difficulty in culturing damaged cells from food samples (Baylis et al., 2000; Garenaux et al., 2008). In a study of pathogenic bacteria in 20 types of vegetables, Bae et al. (2011) showed that samples

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positive for Campylobacter according to a selective agar isolation method were negative when assessed with real-time polymerase chain reaction (PCR) and 16S rRNA sequencing. These results suggest that the use of a selective agar isolation method permits the growth of other microorganisms (non-Campylobacter) and generates falsepositive (non-Campylobacter) results for the detection of Campylobacter spp. in food. The presence of non-Campylobacter contaminants such as Acinetobacter baumannii, Ochrobactrum spp., Pseudomonas spp., and Escherichia coli in agar can greatly complicate the detection and enumeration of Campylobacter by using selective media (Baylis et al., 2000; Line et al., 2008). In particular, extended-spectrum β-lactamase (ESBL)-producing E. coli may be over grown on cefoperazone-based Campylobacter selective media such as Bolton agar, modified charcoal cefoperazone deoxycholate agar (mCCDA), Campy-Cefex agar, and Karmali agar (Chon et al., 2013a; Corry et al., 1995; Jasson et al., 2009; Moran et al., 2011). However, the use of excessive antibiotic agents to inhibit the growth of non-Campylobacter species may impair the growth of sub-lethally injured Campylobacter spp. A balance must be obtained, in which the selective media does not significantly affect the recovery of target microorganisms, but inhibits the growth of undesired competitors on the plate (Line et al., 2008). Because the use of selective media isolation methods for the detection of Campylobacter spp. in fresh produce has been associated with high rates of false-positive results, the development of highly selective media is required. Accordingly, the current study was conducted to develop a highly selective medium for the detection of Campylobacter spp. in food, including fresh produce.

2. Materials and methods 2.1. Bacterial strains and culture conditions Eight strains of C. jejuni (ATCC 33291, 4 human isolates, 1 beef isolate, 1 pork isolate, and 1 chicken isolate) and 3 strains of C. coli (chicken isolates) were obtained from the Chung-Ang University (Anseong-si, Korea), Konkuk University (Seoul, Korea), and National Veterinary Research and Quarantine Service (Anyang-si, Korea) bacterial culture collections. Non-Campylobacter strains (n = 49) isolated from fresh produce using a selective agar isolation method were obtained from the Chung-Ang University bacterial culture collection (Bae et al., 2011). All strains were maintained at − 80 °C in 20% glycerol and were activated by cultivation in blood-free Bolton broth (Oxoid, Basingstoke, UK) with Bolton broth selective supplement (Oxoid). The enrichment broth was microaerobically incubated using a CampyGen gas pack (Oxoid) in an anaerobic jar (Difco Laboratories, Detroit, MI, USA) at 37 °C for 48 h before use. Some false-positive Campylobacter strains were identified using a 16S rRNA sequencing (Solgent Co. Ltd., Daejeon, Korea). Pathogenic bacterial strains (n = 12; E. coli O157:H7 ATCC 35150, E. coli ATCC 25922, Bacillus cereus ATCC 10876, Citrobacter freundi ATCC 8090, Hafnia alvei ATCC 29927, Staphylococcus aureus ATCC 49444, Salmonella enterica ATCC 13076, Salmonella serovar Typhimurium ATCC 19585, Pseudomonas aeruginosa ATCC 15692, P. aeruginosa ATCC 10145, Listeria monocytogenes ATCC 19115, and Cronobacter sakazakii ATCC 12868) and Lactobacillus acidophilus ATCC 04356 were obtained from the Chung-Ang University bacterial culture collection. Spoilage bacterial strains isolated from fresh produce (n = 15; Chryseobacterium balustinum (2), Enterobacter spp. (2), Pantoea agglomerans (1, 2), Bacillus pumilus, Clavibacter michiganensis, Pseudomonas fluorescens (1, 2), Acinetobacter calcoaceticus (1, 2), Stenortophomonas maltophilia (1, 2), Klebsiella pneumoniae, Dickeya zeae, and Pectobacterium carotovorum subsp. carotovorum Pcc21) were obtained from the Rural Development Administration (Suwon-si, Korea). All strains were maintained at −80 °C in 20% glycerol and were activated by cultivation in tryptic soy broth (TSB; Difco) at 37 °C for 24 h before use.

2.2. Comparison of Campylobacter selective media Bolton agar (Oxoid), Hunt agar (MB Cell, Seoul, Korea), mCCDA (Oxoid), and Preston agar (Oxoid) were prepared according to the manufacturer's recommendations. The four selective agars were sterilized by autoclaving at 121 °C for 15 min, and then cooled to 50 °C. After cooling, supplements and blood were gently mixed with basal media and then poured into sterile Petri dishes. Two strains of C. jejuni (ATCC 33291 and 1 human isolate), 3 strains of C. coli (chicken isolates) and 49 strains of non-Campylobacter from fresh produce were cultured individually in blood-free Bolton broth under microaerobic conditions in an anaerobic jar at 42 °C for 48 h. Other indicator bacteria (n = 28; 15 spoilage bacteria, 12 pathogenic bacteria, and 1 lactic acid bacteria) were cultured individually in TSB at 37 °C for 24–48 h. After incubation for 24–48 h, cultured samples were streaked using sterile disposable 10 μL loops onto Bolton agar, Hunt agar, mCCDA, or Preston agar. The plates were incubated under microaerobic conditions at 42 °C for 48 h in an anaerobic jar. Plates containing colonies with a shiny, smooth, round, small, gray, and convex appearance were considered positive for Campylobacter spp.

2.3. Comparison of Bolton agar and mCCDA supplemented with antibiotics Campylobacter strains (n = 7) and non-Campylobacter strains (n = 44) from fresh produce were cultured individually in blood-free Bolton broth under microaerobic conditions in an anaerobic jar at 42 °C for 48 h. Following enrichment, cultures were streaked using s sterile disposable 10 μL loop onto Bolton agar or mCCDA supplemented with the following antibiotics: rifampicin (10 mg/L; Sigma-Aldrich, St. Louis, Mo., USA), polymyxin B (5000 IU/L or 20,000 IU/L; Sigma-Aldrich), sodium metabisulfite (250 mg/L; Sigma-Aldrich), sodium pyruvate (250 mg/L; Sigma-Aldrich), and ferrous sulfate (250 mg/L; SigmaAldrich). Antibiotics were prepared according to the manufacturer's recommendations and were added to the selective media after cooling at 50 °C. The plates were incubated under microaerobic conditions at 42 °C for 48 h.

2.4. Bacterial sensitivity test with antibiotics on Bolton agar supplemented with rifampicin (BR agar) The disk diffusion method was used to determine the antibiotic resistance of the test bacteria. Campylobacter strains (n = 8; 5 C. jejuni and 3 C. coli) and 7 strains of non-Campylobacter (i.e., strains that continued to grow on Bolton agar supplemented with rifampicin) were cultured in blood-free Bolton broth under microaerobic conditions at 42 °C for 48 h. Soft agar containing 0.75% Bacto-agar (Difco) in Bolton broth were autoclaved at 121 °C for 15 min. The sterilized Bolton agar was tempered in a water bath at 50 °C after autoclaving. After cooling, lysed horse blood, Bolton supplement, and rifampicin (10 mg/L) were added to the agar, which was poured into sterile Petri dishes. One hundred microliters of bacterial culture was then inoculated onto Bolton agar supplemented with rifampicin (10 mL) and the plates were dried for 20 min in a safety cabinet. After the agar solidified, antibiotic disks (Sensi-Disc; Difco) were placed on the agar surface and incubated at 42 °C for 48 h under microaerobic conditions by using a CampyGen gas pack in an airtight container (AnaeroPack Rectangular Jar; Mitsubishi Gas Chemical Company, Inc., Tokyo, Japan). Following incubation, the diameter of the inhibition zone was measured. The SensiDiscs contained the following amounts of antibiotics: ampicillin (AM), 10 μg; amikacin (AN) 30 μg; chloramphenicol (C) 30 μg; cephalothin (CF) 30 μg; cefoperazone (CFP) 75 μg; kanamycin (K) 30 μg; gentamicin (GM) 10 μg; tobramycin (NN) 10 μg; penicillin (P) 10 IU; streptomycin (S) 10 μg; sulfamethoxazole–trimethoprim (SXT) 23.75 and 1.25 μg; oxytetracycline (T) 30 μg; and vancomycin (VA) 30 μg.

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2.5. Growth on BR agar supplemented with various concentrations of antibiotics Bolton agar supplemented with rifampicin (BR agar) was shown to have high selectivity for Campylobacter, and accordingly, was used as the base medium. The antibiotics used were sulfamethoxazole (Sigma-Aldrich), trimethoprim (Sigma-Aldrich), or a combination of sulfamethoxazole–trimethoprim. Antibiotics were prepared according to the manufacturer's recommendations at concentrations of 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L, and 500 mg/L. The ratio of the concentration of sulfamethoxazole to trimethoprim was 19:1. Eight strains of Campylobacter (5 C. jejuni and 3 C. coli) and 8 non-Campylobacter strains were cultured in blood-free Bolton broth. All enrichments were incubated under microaerobic conditions by using a CampyGen gas pack in an airtight container at 42 °C for 48 h. Enrichment cultures were then streaked using sterile, disposable 10 μL loops onto BR agar supplemented with sulfamethoxazole, trimethoprim, or sulfamethoxazole–trimethoprim (BRS, BRT, and BRST agar, respectively). Plates were incubated under microaerobic conditions at 42 °C for 48 h in an airtight container. 2.6. Effect of blood addition To investigate the effect of blood, 8 strains of Campylobacter (5 C. jejuni and 3 C. coli) and 8 strains of non-Campylobacter isolated from fresh produce were cultured in blood-free Bolton broth. All enrichments were incubated at 42 °C for 48 h under microaerobic conditions by using a CampyGen gas pack in an airtight container. Enrichment cultures were streaked using a sterile disposable 10 μL loops onto mCCDA, Bolton agar, BR agar, and BRS agar (BR agar containing 50 mg/L sulfamethoxazole) with or without the addition of lysed horse blood. The cultures were incubated at 42 °C for 48 h under microaerobic conditions in an airtight container. 2.7. Validation of selective media for the detection of Campylobacter spp. from chicken and fresh produce Chickens (n = 8, including legs and wings with skin on) and vegetables (n = 50, including bellflower root, cabbage, carrot, chicory, Chinese cabbage, cryptotaenia, cucumber, eggplant, iceberg lettuce, Korean leek, leaf beet, lotus root, mungbean sprouts, organic romaine lettuce, organic Chinese cabbage, pak choi, perilla leaf, radish sprouts, red cabbage, romaine lettuce, spinach, spring onion, sprouts, soybean sprouts, sweet pepper, water dropwort, and zucchini) were purchased from a local grocery store in Anseong-si, Korea. According to the Food and Drug Administration's Bacteriological Analytical Manual procedure (Hunt et al., 2001), food samples (chicken 25 g, vegetable 50 g) were placed in a sterilized stomacher bag containing 100 mL of blood-free Bolton broth. After stomaching for 5 min, homogenized samples were enriched at 42 °C for 48 h under microaerobic conditions by using a CampyGen gas pack in an airtight container. Enrichment cultures were streaked using a sterile disposable 10 μL loops onto mCCDA agar, BR agar, BRS agar, and BRSC agar (BRS agar containing 0.5 mg/L ciprofloxacin [Sigma-Aldrich]). Plates were incubated under microaerobic conditions by using a CampyGen gas pack in an airtight container at 42 °C for 48 h. 2.8. Identification of isolates from chicken and fresh produce Strains that exhibited continuous growth on BRS agar (n = 15) were identified using a conventional PCR assay with MJ Mini™ Gradient Thermal Cycler (Bio-Rad, Foster City, USA), to confirm their identity as Campylobacter spp. The forward primer was 5′-AAT CTA ATG GCT TAA CCA TTA-3′, and the sequence of the reverse primer was 5′-GTA ACT AGT TTA GTA TTC CGG-3′ (Linton et al., 1997). This assay has been tested against strains of all species in the genus Campylobacter (rrs gene).

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Isolates were identified using the API 20E kit (BioMérieux, Marcy l'Etoile, France), according to the manufacturer's instructions, and the results were analyzed using apiweb™ (https://apiweb.biomerieux. com). To confirm the API 20E results, selected strains were identified using the 16S rRNA sequencing, 16S rRNA sequencing was requested to the specialized company (Solgent Co. Ltd, Daejeon, Korea). Briefly, DNA isolation was performed using the purification bead (Solgent). The universal primer 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′) were used to amplify community 16S rRNA genes. Each PCR mixture made using Solg™ EF-Taq DNA Polymerase (Solgent) according to the manufacturer's instructions. PCR amplification was performed using GeneAmp® PCR system 9700 (Applied Biosystem, Foster City, USA) with one denaturation step at 95 °C for 10 min followed by 30 cycles of 95 °C for 20 s, 50 °C for 40 s, and 72 °C for 90 s with a final elongation step at 72 °C for 10 min. PCR products were purified using a Solg™ PCR purification kit (Solgent) according to the manufacturer's instructions, and the purified bacterial amplicons were cloned using a T-Blunt™ PCR Cloning Kit (Solgent) following the manufacturer's instructions. Clone sequences were determined by using a ABI 3730XL DNA Sequencer (Applied Biosystem). Close relative and phylogenetic affiliation of the sequence were checked using the BLAST search (best hit with similarity, ≥97%) at the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih. gov/).

3. Results 3.1. Comparison of Campylobacter selective media We investigated the selectivity of Campylobacter selective media by using 5 Campylobacter spp. (3 C. jejuni and 2 C. coli) and 49 nonCampylobacter strains from fresh produce (Table 1). All Campylobacter spp. grew on both Bolton agar and mCCDA, whereas only 1 strain of C. jejuni grew on Preston agar, and no strains grew on Hunt agar. Of the 49 non-Campylobacter strains, 39 strains grew on mCCDA, 37 grew on Bolton agar, 4 grew on Preston agar, and 2 grew on Hunt agar. Hunt agar and Preston agar inhibited the growth of most nonCampylobacter strains and all C. jejuni and C. coli strains tested. These results indicate that while Bolton agar and mCCDA are more suitable selective media for Campylobacter spp. than are Hunt agar and Preston agar, their selectivity of Campylobacter spp. needs improvement. Next, we used 16S rRNA sequencing to identify selected nonCampylobacter strains that were isolated from fresh produce with selective agar isolation methods but grew on Campylobacter selective medium (Table 2). Of the 13 strains assessed, 5 were Acinetobacter spp., 3 were Pseudomonas spp., 3 were Ochrobactrum intermedium, 1 was Brevundimonas diminuta, and 1 was Myroides odoratimimus. Given these results, Acinetobacter spp., Pseudomonas spp., and O. intermedium in fresh produce were deemed recoverable on Campylobacter selective media such as Bolton agar and mCCDA. Moreover, colonies of these strains were not easily distinguishable from Campylobacter, thereby generating false-positive results (data not shown). Further, 28 additional strains of indicator bacteria (15 spoilage bacteria, 12 pathogenic bacteria, and 1 lactic acid bacterium) were tested for growth on Campylobacter selective media under microaerobic conditions (Table 1). Two strains of P. aeruginosa grew on Bolton agar and Preston agar, and P. fluorescence-2 grew on Bolton agar and mCCDA. None of the strains tested grew on Hunt agar. Of the spoilage bacteria isolated from fresh produce, 2 strains of A. calcoaceticus grew on mCCDA and A. calcoaceticus-2 grew on Bolton agar. Because Pseudomonas colonies are similar in appearance to Campylobacter colonies, we suggest that Pseudomonas spp. presents a problem for the detection of Campylobacter (data not shown).

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Table 1 Growth of Campylobacter spp. and indicator microorganism on 4 selective media under microaerobic condition. Strains

Table 1 (continued) Strains

Selective agar

Selective agar Bolton agar

Hunt agar

mCCDA

Preston agar

1 C. jejuni 2 C. coli 3 C. coli 4 C. coli 11 C. jejuni ATCC 33291

+++a +++ +++ + +++

− − − ––

+++ +++ +++ + +++

++ − − – –

12 Hot pepper-1 13 Dropwort-1 14 Cucumber-1 15 Cucumber-2 16 Garlic stalk-1 17 Head lettuce-1 18 Cabbage-1 19 Sprout-1 20 Cucumber-3 21 Green bean sprout-1 22 Dropwort-2 23 Organic lettuce-1 24 Cucumber-4 25 Balloon flower root-2 26 Balloon flower root-1 27 Bean sprout-1 28 Bean sprout-2 29 Bean sprout-3 30 Green bean sprout-2 31 Green bean sprout-3 32 Green bean sprout-4 33 Garlic stalk-2 34 Onion-1 35 Onion-2 36 Sprout-2 37 Sprout-3 38 Paprika 39 Red cabbage-1 40 Red cabbage-2 41 Cabbage-2 42 Dropwort-2 43 Carrot-1 44 Korean leek-1 45 Korean leek-2 46 Chinese cabbage-1 47 Chinese cabbage-2 48 Chinese cabbage-3 49 Organic sesame leaf-1 50 Organic sesame leaf-2 51 Sesame leaf-1 52 Sesame leaf-2 53 Sesame leaf-3 54 Sesame leaf-4 55 Organic lettuce-2 56 Organic lettuce-3 57 Lettuce-1 58 Lettuce-2 59 Lettuce-3 60 Lettuce-4

+++ ++b +++ + − +++ ++ ++ +++ +++ − +++ − +++ +++ +++ +++ +++ − +++ ++ ++ +++ +++ +++ +++ ++ − +++ +++ +++ − +++b +++ − + +++ − +b ++ + − +++ +++ +++ − − +++ +++

− − − − − − − − − − − − − − − − − − − − − − ++b − − − − − − − − − +++b − − − − − − − − − − − − − − − −

+++ + +++ ++ + +++ ++ ++ +++ +++ − +++ − +++ +++ +++ +++ +++ − +++ ++ ++ +++ +++b +++ +++ ++ − +++ +++ +++ − +++ +++ − + +++ − ++ ++ + − +++ +++ +++ − − +++ +++

− − ++ − − − − − − − − − − − − − − − − − − + − − − − − + − − − + − − − − − − − − − − − − − − − −

Escherichia coli O157:H7 ATCC 35150 Escherichia coli ATCC 25922 Bacillus cereus ATCC 10876 Citrobacter freundii ATCC 8090 Hafnia alvei ATCC 29927 Staphylococcus aureus ATCC 49444 Salmonella enterica ATCC 13076 Salmonella Typhimurium ATCC 19585 Pseudomonas aeruginosa ATCC 15692 Pseudomonas aeruginosa ATCC 10145 Listeria monocytogenes ATCC 19115 Cronobacter sakazakii ATCC 12868 Lactobacillus acidophilus ATCC 04356

− − − − − − − − + + − − −

− − − − − − − − − − − − −

− − − − − − − − − − − − −

− − − − − − − − + + − − +

Chryseobacterium balustinum-2 Enterobacter sp.-2 Pantoea agglomerans-1

− − −

− − −

− − −

− − −

Pantoea agglomerans-2 Bacillus pumilus Clavibacter michiganensis Pseudomonas fluorescens-1 Pseudomonas fluorescens-2 Acinetobacter clacoaceticus-1 Acinetobacter clacoaceticus-2 Stenotrophomonas maltophilia-1 Stenotrophomonas maltophilia-2 Klebsiella pneumonia Dickeya zeae Pectobacterium carotovorum subsp.

Bolton agar

Hunt agar

mCCDA

Preston agar

− − − − +++ − + − − − − −

− − − − − − − − − − − −

+ − − − + +++ +++ − − − − −

− − − − − − − − − − − +

a +, positive; −, negative; ++, two positive in three experiments; +++, three positive in three experiments. b Colony morphology was very similar to that of Campylobacter spp.

3.2. Comparison of Bolton agar and mCCDA supplemented with antibiotics To suppress the growth of non-Campylobacter, strains were grown on Bolton and mCCDA supplemented with a variety of antibiotics (Table 3), with conventional Bolton agar and mCCDA used as controls. Bolton agar supplemented with rifampicin (10 mg/L) inhibited the growth of 36 strains of non-Campylobacter, but did not inhibit all Campylobacter strains. Bolton agar supplemented with polymyxin B (20,000 IU/L) inhibited the growth of 36 strains of non-Campylobacter and 4 strains of Campylobacter. The addition of various antibiotics to mCCDA almost completely failed to inhibit the growth of nonCampylobacter. On the basis of these results, we concluded that the selectivity of BR agar for Campylobacter spp. was higher than that of mCCDA containing any of the 5 antibiotics tested.

3.3. Bacterial sensitivity test with antibiotics on BR agar In the experiments described above, BR agar exhibited high selectivity for Campylobacter spp.; however, non-Campylobacter strains nevertheless grew on BR agar. To inhibit the growth of these bacteria, we tested their sensitivity to antibiotics by using the agar disk diffusion test. Table 4 shows the inhibition zone diameters of the antibiotics on BR agar. Although gentamicin and amikacin inhibited the growth of all non-Campylobacter strains tested, they also inhibited all Campylobacter spp. tested. Cefoperazone and sulfamethoxazole–trimethoprim inhibited 6 and 3 of the 7 non-Campylobacter strains tested, respectively. However, of the 8 strains of Campylobacter tested, cefoperazone inhibited 2 strains and sulfamethoxazole–trimethoprim inhibited Table 2 16S rRNA sequence-based identification of non-Campylobacter species isolated from fresh produce that grew on 4 selective media. Strains

16S rRNA sequencing

15 Cucumber-2 16 Garlic stalk-1 18 Cabbage-1 21 Green bean sprout-1 23 Organic lettuce-1 31 Green bean sprout-3 34 Onion-1 35 Onion-2 36 Sprout-2 37 Sprout-3 40 Red cabbage-2 44 Korean leek-1 48 Chinese cabbage-3

Brevundimonas diminuta Pseudomonas putida Acinetobacter sp. Myroides odoratimimus Ochrobactrum intermedium Acinetobacter sp. Ochrobactrum intermedium Pseudomonas syringae Pseudomonas sp. Acinetobacter calcoaceticus Acinetobacter sp. Ochrobactrum intermedium Acinetobacter calcoaceticus

Identity (%) 99 99 99 100 100 100 100 97 99 100 100 100 99

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Table 3 Comparison of Bolton agar and mCCDA supplemented with various antibiotics for the detection of Campylobacter spp. and non-Campylobacter strains. Straina

Bolton agar mCCDA a b c

Antibiotics

Campy (n = 7) Non-Campy (n = 44) Campy (n = 7) Non-Campy (n = 44)

Controlb

Rifampicin (10 mg/L)

Polymyxin B (5000 IU/L)

Polymyxin B (20,000 IU/L)

Sodium metabisulfite (250 mg/L)

Sodium pyruvate (250 mg/L)

Ferrous sulfate (250 mg/L)

7 (100)c 40 (91) 7 (100) 42 (95)

7 (100) 8 (18) 7 (100) 36 (82)

7 (100) 33 (75) 7 (100) 40 (91)

3 (43) 8 (18) 7 (100) 41 (93)

3 (43) 40 (91) 7 (100) 41 (93)

7 (100) 40 (91) 7 (100) 41 (93)

7 (100) 40 (91) 7 (100) 39 (89)

Campy, Campylobacter strains; Non-Campy, non-Campylobacter strains isolated from fresh produce. Control, Bolton agar or mCCDA. Numbers of positive strains (%).

none. Accordingly, sulfamethoxazole–trimethoprim was chosen as the second antibiotic for inclusion in BR agar. 3.4. Growth on BR agar supplemented with antibiotics at various concentrations To increase the selectivity of BR agar for Campylobacter spp., we determined the optimal concentrations of sulfamethoxazole, trimethoprim, or sulfamethoxazole–trimethoprim in BR agar. Table 5 shows the growth of Campylobacter spp. and non-Campylobacter on BR agar supplemented with 2 individual antibiotics or the antibiotic mixture at concentrations of 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L, and 500 mg/L. BR agar supplemented with 50 mg/L sulfamethoxazole inhibits the growth of 2 strains of non-Campylobacter but did not inhibited the growth of any of the strains of Campylobacter spp. tested. Given these results, we designed BRS agar as BR agar containing 50 mg/L sulfamethoxazole. It should be noted that certain strains grew continuously on BRS agar, and their colonies were not easily distinguishable (Fig. 1). 3.5. Effect of the addition of blood We examined the effect of the addition of blood on the growth of Campylobacter spp. and non-Campylobacter strains. We found no difference in the growth of Campylobacter spp. and non-Campylobacter strains on Bolton agar containing blood relative to growth on normal Bolton agar (Table 6). Although 2 strains of Campylobacter grew more weakly on blood-free BRS agar than on BRS agar containing blood, the difference was negligible. These results indicate that

blood does not affect the growth of Campylobacter on the selective medium. 3.6. Validation of the selective media Table 7 shows the number of Campylobacter-positive samples from chickens (n = 8) and fresh produce (n = 50) on mCCDA (control), BR agar, BRS agar, and Bolton agar supplemented rifampicin, sulfamethoxazole, and ciprofloxacin (BRSC agar). On mCCDA, all of the chicken samples were positive and 28 (56%) of the fresh produce samples were positive. All of chicken samples were positive on BR agar and BRS agar, and 13 (26%) and 9 (18%) fresh produce samples were positive on BR agar and BRS agar, respectively. Because numerous studies have shown that ciprofloxacin inhibits the growth of Pseudomonas spp., we examined the effect of the addition of ciprofloxacin to BRSC agar on growth. On BRSC agar, 7 of 8 chicken samples were positive and 6 positive results were obtained from these 9 positive samples from fresh produces. With the progressive addition of antibiotics, the growth of isolates from fresh produce was increasingly inhibited, although the growth of chicken isolates was not inhibited on any media except BRSC agar. PCR analysis identified the 15 strains that grew continuously on BRS agar as false-positive Campylobacter. These data indicated that BRS agar inhibited certainly on-Campylobacter from fresh produce samples, but failed to inhibit any of the false-positive strains from chicken samples. On the basis of the API kit results, the isolates from chicken and fresh produce were tentatively identified as E. coli, Enterobacter spp., Klebsiella spp., and Pseudomonas spp., although the matching scores were generally low (data not shown). To confirm the API kit results, selected isolates were unambiguously identified using 16S rRNA

Table 4 Inhibition zone diameter (cm) of antibiotics on BR agar (Bolton agar supplemented with rifampicin at 10 mg/L). Strains

Antibiotics AMa

AN

C

CF

CFP

K

GM

NN

P

S

SXT

T

VA

2 8 9 10 11 3 4 6

C. jejuni C. coli C. coli C. coli C. jejuni ATCC 33291 C. jejuni C. jejuni C. jejuni

0.0 2.0 2.2 2.1 2.0 NDb ND ND

3.1 3.2 3.2 3.0 3.3 ND ND ND

3.6 3.6 4.4 3.9 3.4 ND ND ND

0.0 1.0 0.0 0.0 0.0 ND ND ND

0.0 0.9 0.0 1.0 0.0 0.0 0.0 0.0

2.7 2.3 0.0 2.9 2.8 ND ND ND

3.8 3.4 3.6 3.2 3.4 ND ND ND

2.5 2.2 2.8 2.0 2.2 ND ND ND

0.0 1.1 0.0 1.3 0.9 ND ND ND

2.8 3.0 0.0 3.2 2.6 ND ND ND

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

1.2 1.4 0.0 1.5 3.6 ND ND ND

0.0 0.0 0.0 0.0 0.0 ND ND ND

23 36 16 20 34 35 38

O. intermedium Pseudomonas sp. P. putida Unidentifiedc O. intermedium P. syringae Unidentified

0.0 0.0 ND ND ND ND ND

2.4 3.0 ND ND ND ND ND

0.0 2.5 ND ND ND ND ND

0.0 0.0 ND ND ND ND ND

2.0 0.0 1.8 1.8 2.6 1.0 1.8

0.0 3.0 ND ND ND ND ND

2.8 2.8 ND ND ND ND ND

0.0 2.4 ND ND ND ND ND

0.0 0.0 ND ND ND ND ND

2.3 0.0 ND ND ND ND ND

2.5 0.0 0.0 2.2 0.0 0.0 3.6

4.4 0.0 ND ND ND ND ND

0.0 0.0 ND ND ND ND ND

a Abbreviations: AM, ampicillin (10 μg); AN, amikacin (30 μg); C, chloramphenicol (30 μg); CF, cephalothin (30 μg); CFP, cefoperazone (75 μg); K, kanamycin (30 μg); GM, gentamicin (10 μg); NN, tobramycin (10 μg); P, penicillin (10 IU); S, streptomycin (10 μg); SXT, sulfamethoxazole–trimethoprim (23.75 μg, 1.25 μg); T, oxytetracycline (30 μg); VA, vancomycin (30 μg). b ND: not determined. c Unidentified strain from API kit and 16S rRNA sequencing method.

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Table 5 Growth of Campylobacter spp. and non-Campylobacter strains isolated from food samples on BR agara supplemented with antibiotics. Strains

Controlb

6.25

12.5

25

50

100

500

6.25

12.5

25

50

100

500

6.25

12.5

25

50

100

500

2 3 4 6 8 9 10 11

C. C. C. C. C. C. C. C.

+d + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + Δ + + + Δ

− − − − − − − −

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + Δ

− − Δ − + + + −

− − − − − − − −

16 20 23 34 35 36 44

P. putida Unidentifiede O. intermedium O. intermedium P. syringae Pseudomonas sp. O. intermedium

+ + + + + + +

+ + − − + + +

+ + − − + + +

+ + − − + + +

+ + − − + + +

+ + − − + + +

ND + − − + − +

+ + + + + + +

+ + + + + + +

+ + + + + + +

+ + + + + + +

+ + + − + + +

ND + − + + + +

ND + + + + + +

ND + − + + + +

ND + − + + + +

ND + Δ Δ + + +

ND + − − + + +

ND Δ − − Δ Δ Δ

a b c d e

jejuni jejuni jejuni jejuni coli coli coli jejuni ATCC 33291

Sulfamethoxazole (mg/L)

Sulfamethoxazole–trimethoprim (mg/L)c

Trimethoprim (mg/L)

BR agar, Bolton agar supplemented with rifampicin (10 mg/L). Control, BR agar. The ratio of the concentration of sulfamethoxazole to trimethoprim was 19:1. +, growth; −, no growth; Δ, growth but weak; ND, not determined. Unidentified strain from API kit and 16S rRNA sequencing method.

sequencing, which confirmed the results obtained from the API kit. Given these data, we conclude that E. coli, Enterobacter spp., Klebsiella spp., and Pseudomonas spp. remain a problem and interfere with the detection of Campylobacter spp. in food. 4. Discussion Although numerous selective media have been described for the detection of Campylobacter spp. in food, the presence of non-Campylobacter contaminants can lead to false-positive results. Moreover, Campylobacter spp. in food have not been accurately detected and enumerated. In a previous study, we determined the frequency of false-positive results during the detection of Campylobacter spp. in fresh produce by using selective media. Subsequently, we developed a selective medium for the detection of Campylobacter spp. in fresh produce. Nicorescu and Crivineany (2009) reported that mCCDA and Preston agar provided a greater selectivity for Campylobacter spp. then did Skirrow agar and Karmali agar. However, in our study, the recovery rates for Campylobacter and non-Campylobacter were much lower with Hunt agar and Preston agar than with Bolton agar and mCCDA. Line and Berrang (2005), in a study on the detection of Campylobacter in broiler carcass rinses, reported that Campy-Line agar provided greater selectivity and supported the growth of fewer contaminants than did Campy-Cefex agar. Oyarzabla et al. (2005) reported that Campy-Cefex agar and its variants yielded the best results for the direct enumeration of Campylobacter spp. from poultry carcass rinses. Campy-Line agar had lower counts than CAMPY agar, mCCDA, and Karmali agar, although the differences were not statistically significant. Many studies on the

A

B

detection of Campylobacter in food have reported that the contaminating bacteria were Pseudomonas spp., E. coli, Acinetobacter spp., and Arcobacter spp. (Chon et al., 2013b; Engberg et al., 2000; Karmali et al., 1986). Similarly, our results showed that these strains were problematic for the detection of Campylobacter on agar. Moreover, colonies of these strains were not easily distinguishable from those of Campylobacter, thus generating false-positive results. Ng et al. (1985) and Goossens et al. (1986) reported that high levels of polymyxin B inhibited C. coli. Interestingly, Chon et al. (2012) reported that supplementation of mCCDA with polymyxin B (100,000 IU/L) effectively eliminated competing microflora from chicken carcass rinses, resulting in higher isolation rates for C. coli and C. jejuni. In this study, mCCDA supplemented with polymyxin B (20,000 IU/L) failed to inhibit most non-Campylobacter strains. Line et al. (2008) reported that modified Campy-Line agar (CLA) containing sulfamethoxazole eradicated contaminants from broiler rinses, resulting in the selective detection and enumeration of Campylobacter spp. colonies on CLA + sulfamethoxazole plates. In this study, BRS agar inhibited O. intermedium, although certain non-Campylobacter strains were not inhibited and their colonies were not easily distinguishable from those of Campylobacter. Despite the addition of antibiotics to selective media, the growth of some competing organisms was not inhibited, which interfered with the accurate detection of Campylobacter. In particular, we found that Pseudomonas isolated from fresh produce was resistant to rifampicin and sulfamethoxazole. Many researchers have reported that the presence of blood on agar does not affect the detection of Campylobacter (Goossens et al., 1986; Williams et al., 2009). Our results are in agreement with these findings.

C

D

Fig. 1. Appearance of colonies on Bolton agar supplemented with rifampicin (10 mg/L) and sulfamethoxazole (50 mg/L) after 48 h at 37 °C under microaerobic incubations (A, Campylobacter jejuni ATCC 33291; B, 16 Pseudomonas putida from garlic stalk-1; C, 35 Pseudomonas syringae from onion-2; D, 36 Pseudomonas sp. from sprout-2).

J.-H. Yoo et al. / International Journal of Food Microbiology 189 (2014) 67–74

73

Table 6 Comparison of selective agars supplemented with various antibiotics and/or blood for the detection of Campylobacter spp. and non-Campylobacter strains. Strain

mCCDA Bolton agar

Bolton agar (−blood)

Bolton agar + Ra

Bolton agar + R (−blood)

Bolton agar + R + S

Bolton agar + R + S (−blood)

2 3 4 6 8 9 10 11

C. jejuni C. jejuni C. jejuni C. jejuni C. coli C. coli C. coli C. jejuni ATCC 33291

+b + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

16 20 23 34 35 36 44

P. putida Unidentifiedc O. intermedium O. intermedium P. syringae Pseudomonas sp. O. intermedium

+ + + + + + +

+ + + + + + +

+ + + + + + +

+ + + + + + +

+ + + + + + +

+ + − − + + +

+ + − − + + +

a b c

Abbreviations: R, rifampicin (10 mg/L); S, sulfamethoxazole (50 mg/L); C, ciprofloxacin (0.5 mg/L). +, growth; −, no growth. Unidentified strain from an API kit and 16S rRNA sequencing method.

During the isolation of Campylobacter from chicken samples, Moran et al. (2011) reported that 93% of the contaminants on mCCDA plates were ESBL-producing E. coli, which suggested that the organisms had the ability to hydrolyze the cefoperazone present in Bolton broth and mCCDA. During the detection of Campylobacter from chicken carcass rinses, Chon et al. (2013a) also reported that the most dominant contaminants were ESBL-producing E. coli, which have been isolated from chickens in the United Kingdom (Warren et al., 2008), the Netherlands (Overdevest et al., 2011), and Portugal (Costa et al., 2010). In this study, the growth of the indicator bacteria E. coli, K. pneumoniae, and Enterobacter spp. was inhibited on Bolton agar, mCCDA, Hunt agar, and Preston agar. However, the isolates from chicken and fresh produce that grew on BRS agar were identified as E. coli, Klebsiella spp., Enterobacter spp., and Pseudomonas spp. We speculate that our results might be due to the fact that the strains isolated from chicken and fresh produce on BRS agar were ESBL-producing bacteria or antibiotic resistant strains. In conclusion, the selective medium BRS agar inhibited the growth of non-Campylobacter from fresh produce to a greater degree than did Bolton agar and mCCDA, without affecting the detection of Campylobacter spp. and with increased selectivity for Campylobacter spp. Some nonCampylobacter strains were still able to grow on BRS agar, suggesting that further studies are required to increase the selectivity of BRS agar and inhibit the growth of Pseudomonas and ESBL-producing bacteria. We suggest that BRS agar is a potential alternative to Bolton agar and mCCDA for the detection of Campylobacter spp. from fresh produce.

Table 7 Number of samples showing positive result when cultured on various Campylobacter selective mediaa. mCCDA Chicken (n = 8) Fresh produce (n = 50)

Bolton Bolton Bolton agar + Rb agar + R + S agar + R + S + C

8 (100)c 8 (100) 28 (56) 13 (26)

8 (100) 9 (18)

7 (88) 6d (12)

a Numbers of food samples prepared according to the Food and Drug Administration's Bacteriological Analytical Manual procedure. b Abbreviations: R, rifampicin (10 mg/L); S, sulfamethoxazole (50 mg/L); C, ciprofloxacin (0.5 mg/L). c Numbers of positive strains (%). d Number of positive results confirmed as non-Campylobacter strains using API kit and 16S rRNA sequencing method.

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