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Journal of Food Protection, Vol. 71, No. 4, 2008, Pages 850–854 Copyright ᮊ, International Association for Food Protection

Research Note A Comparison of Media for the Isolation of Arcobacter spp. from Retail Packs of Beef

SARAH HAMILL,1 SIDNEY D. NEILL,1,2 AND ROBERT H. MADDEN1,3*

1Department of Food Science, Queen’s University of Belfast, Newforge Lane, Belfast BT9 5PX, Northern Ireland; and 2AESD and 3Food Microbiology Branch, Agri-Food & Biosciences Institute, Newforge Lane, Belfast BT9 5PX, Northern Ireland Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/4/850/1682209/0362-028x-71_4_850.pdf by guest on 27 September 2021 MS 07-524: Received 1 October 2007/Accepted 25 November 2007

ABSTRACT

In order to determine the most effective protocol for the isolation of Arcobacter spp. from retail packs of beef, three published methods (A, B, and C) were selected. In addition, a modified version of method B was studied (method D). The ability of the four methods to isolate Arcobacter from standardized beef samples (n ϭ 80) was compared with presumptive Arcobacter isolates being identified to and species level, using multiplex PCR methods. The presence of Arcobacter in enrichment broths was also investigated using PCR techniques. Overall, the modified enrichment and selection media of Johnson and Murano (method D) gave the highest recovery of Arcobacter. Recovery using these media was enhanced by incubating the enrichment and selection media in a microaerobic cabinet rather than air, and the inclusion of streaking the enrichment broth onto selective agar after 24 h in addition to 48 h. Method D yielded significantly more Arcobacter-positive samples of beef (P Ͻ 0.01) than did the three other methods investigated.

Increasingly, members of the genus Arcobacter are be- ination in beef ranges from 0 to 34% (19, 30). Differences ing recognized as potential human pathogens. Of the six in the isolation methods used may contribute to the varia- Arcobacter species described to date, A. butzleri, A. cryae- tion observed. As part of a study into the prevalence of rophilus, and A. skirrowii have been associated with various arcobacters in retail packs of red meats on the island of human illnesses, most frequently gastroenteritis (10, 20, 31, Ireland, an effective Arcobacter isolation protocol was re- 32), but also bacteremia (15, 24, 34). However, due to their quired. The aim of this study was to compare four isolation similarity to spp., the true prevalence of methods in order to identify the most effective method for gastroenteritis caused by arcobacters may be underestimat- the isolation of Arcobacter species from retail packs of raw ed (25, 33). beef. Arcobacter species contaminate a wide range of human food products, having been isolated from chicken, pork, MATERIALS AND METHODS beef, turkey, and duck (1, 3, 5, 8, 23, 27, 35). They have Atmosphere. Microaerobic incubations were performed in a also been isolated from a variety of water sources, including Ruskin Concept 400 cabinet (Ruskin Technology, Ltd., Leeds, drinking water (2, 9, 16, 26). The frequent association of UK) set to give an atmosphere (vol/vol) of 10% CO2,5%O2,4% arcobacters with products commonly consumed by humans H2, and the balance being N2. may indicate that the ingestion of contaminated foods or Media. All media were obtained from Oxoid, Basingstoke, water is a major route of transmission of these potentially UK, and all chemicals from Sigma, Poole, UK, unless otherwise pathogenic enteric microbes. stated. Several media have been described for the detection of Arcobacter spp. in foodstuffs, with most being developed Sample collection. Retail packs of raw beef braising steak using poultry meat as a substrate, but currently there are (n ϭ 40) and raw ground beef (n ϭ 40) were purchased from no standardized methods. The isolation procedures are gen- several supermarkets and butchers premises in Belfast, Northern erally dependent on the use of enrichment media containing Ireland. Where possible, different ‘‘use by’’ dates and EU proces- several antibiotics, which act as selective supplements (4, sor numbers were selected to ensure a variety of sources was sampled. Pack sizes ranged from 300 to 500 g. In all cases, sample 12, 17, 18, 21), although some methods also rely on mo- processing was initiated within 4 h of purchase. tility, i.e., their ability to pass through membrane filters as a selective measure (8). Sample preparation. Samples (60 g) were aseptically re- The reported prevalence of arcobacters in foodstuffs moved from retail packs using sterile, disposable scalpels (Swann differs widely among studies, for example reported contam- Morton, Sheffield, UK) and forceps (Steriseal Forceps, Redditch, UK), added to 60 ml of peptone water (Lab104, LabM, Bury, * Author for correspondence. Tel: 44 28 90255312; Fax: 44 28 90668376; UK), and homogenized in a stomacher blender (Seward 400, Sew- E-mail: [email protected]. ard, Ltd., London, UK) at full power (1 min). The homogenate J. Food Prot., Vol. 71, No. 4 ISOLATION OF ARCOBACTER FROM BEEF 851

TABLE 1. Basis of the four isolation methods compared for their ability to isolate Arcobacter spp. from retail packs of raw beef in Northern Irelanda Supplements used and methodological details for each methodb

Enrichment broth Solid medium

Method Formulation Incubation conditions Plating medium Plate incubation conditions

-A. Houf et al. (13) Arcobacter broth: 30؇C, 48 h, microaero- Arcobacter plating me- 30ЊC, microaerobic; ex 16 mg of cefoperazone bicc diumd: amine at 24 h, 48 h, 10 mg of amphotericin B 16 mg of cefoperazone 72 h, 120 h 100 mg of 5-flourouracil 10 mg of amphotericin B 32 mg of novobiocin 100 mg of 5-flourouracil 64 mg of trimethoprim 32 mg of novobiocin

5% (vol/vol) lysed horse 64 mg of trimethoprim Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/4/850/1682209/0362-028x-71_4_850.pdf by guest on 27 September 2021 blood B. Johnson and Murano JM broth: 30ЊC, 24 h, 48 h, aero- JM plating medium: 30ЊC, aerobic; examine (17, 18) 32 mg of cefoperazone bic 32 mg of cefoperazone at 48 h 200 mg of 5-flourouracil 5% (vol/vol) defibrinat- 3% activated charcoal ed sheep blood 0.25% bile salts C. Atabay and Corry Enrichment broth: 25ЊC, 48 h, aerobic Enrichment broth: 25ЊC, aerobic; examine (4) Arcobacter broth (Oxoid Arcobacter broth (Oxoid at 48 h CM965) plus CAT CM965) plus CAT supplement (Oxoid supplement (Oxoid SR174E) SR174E) plus 2% (wt/vol) agar number 1 (Oxoid) D. Johnson and Mura- JM broth: as per meth- 30ЊC, 24 h,48h,mi- JM plating medium: as 30ЊC, microaerobicc; ex- no, modified (17, 18) od B croaerobicc per method B amine at 48 h a Items in bold are modified from the original method published. b Given quantities of selective agents are per liter of each formulation. c Details of microaerobic atmospheres are given in the text. d Loop inoculation was used in all cases: 10 ␮l of enrichment broth streaked directly onto the appropriate plating medium. comprised a standardized sample material to be used for the com- of DNA, a sacrificial sampling scheme was used with enrichment parison of methods. broths for methods B and D. Therefore, duplicate enrichment broths were prepared from the samples, which were prepared as Arcobacter spp. isolation procedures. Four isolation meth- noted above to ensure uniformity. After enrichment broths had ods (Table 1) were selected for comparison, and a sacrificial plate- been streaked onto selective media, samples of broth culture (4 sampling protocol was used with all methods to facilitate exam- ml) were transferred to plastic universal bottles (Bibby Sterilin, ination of plates and picking of suspect colonies. A standardized Ltd., Staffordshire, UK) and stored (Ϫ20ЊC) until required. sample (10 g) was added to 90 ml of enrichment broth in all cases. Positive controls consisted of A. butzleri NCTC 12481, A. The following modifications to the methods were applied: skirrowii NCTC 12713, and A. cryaerophilus CCUG 17802. (i) Method A (13). Incubation at 30ЊC was used, instead of These were also used in the phenotyping and genotyping studies 28ЊC. Samples from broths incubated for 48 h were streaked onto described below. four selective agar plates for examination at 24, 48, 72, and 120 h. Plates were discarded after the selection of suspect colonies. Phenotypic characterization. Suspect Arcobacter isolates Note that examination after 120 h was an addition to the published were characterized using the hanging-drop method for motility protocol. and by Gram stain reaction and morphology (7). Cultures (ap- (ii) Method B (17, 18). As modified (29) by the inclusion of proximately 24 h, 30ЊC) grown on blood agar containing 5% (vol/ an additional sampling of the enrichment broth at 24 h; hence, vol) defibrinated horse blood were used for analysis. two plates were incubated per sample (iii) Method C (4). Used as published, utilizing one plate per Genotypic identification of genus and species. Suspect Ar- sample cobacter colonies were purified by streaking onto blood agar (iv) Method D. This used the protocol of method B, but all plates. Using a 10-␮l loop, a sweep of cells was transferred to 1 media were incubated in a microaerobic cabinet. Enrichment ml of buffer (150 mM NaCl, 15 mM EDTA, and 10 mM Tris- broths were incubated with the caps loose. HCl; pH 8.0) for DNA extraction. The remaining growth was Due to the scale of the study (with 720 plates of selective harvested into cryovials containing 1 ml of nutrient broth supple- agar requiring examination), where growth was obtained only one mented with 10% (vol/vol) glycerol and stored at Ϫ80ЊC. suspect colony per plate, based on its morphology, was picked for Phenol/chloroform extraction was used to obtain DNA from subsequent purification, phenotyping, and genotyping. To facili- pure cultures (11). Puregene genomic purification kits (Gentra tate the sampling of enrichment broths for subsequent extraction Systems, Inc., Minneapolis, Minn.), used according to manufac- 852 HAMILL ET AL. J. Food Prot., Vol. 71, No. 4

TABLE 2. Primers used in this studya Primer Sequence Target and expected product size

U1 5Ј-CAG CMG CCG CGG TAA TWC Bacterial DNA U2 5Ј-CCG TCA ATT CMT TTR AGT TT U1 ϩ U2 ϭ 408 bp Arco 1A 5Ј-GTC GTG CCA AGA AAA GCC A Arcobacter genus Arco 1B 5Ј-TTC GCT TGC GCT GAC AT Arco 1A ϩ Arco 1B ϭ 331 bp Arco 5Ј-CGT ATT CAC CGT AGC ATA GC Arcobacter species Butz 5Ј-CCT GGA CTT GAC ATA GTA AGA ATG A Arco ϩ Butz ϭ 401-bp Skir 5Ј-GGC GAT TTA CTG GAA CAC A Arco ϩ Skir ϭ 614-bp Cry 1 5Ј-TGC TGG AGC GGA TAG AAG TA Cry 1 ϩ Cry 2 ϭ 257-bp Arcobacter cryaerophilus Cry 2 5Ј-AAC AAC CTA CGT CCT TCG AC Cry 1 ϩ Cry 2 ϭ 257-bp Arcobacter cryaerophilus a The bacterial DNA primers were described by Lawrence and Gilmour (22), the genus-specific primers by Bastyns et al. (6), and the species-specific primers by Houf et al. (14). Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/4/850/1682209/0362-028x-71_4_850.pdf by guest on 27 September 2021 turing instructions for 1-ml volumes containing gram-negative (21 of 24), with the remaining three being A. cryaerophilus. , were used to obtain DNA from enrichment broths. No isolates were positive for only the genus primer. DNA from isolates and enrichment broths was subjected to During storage, broths from 10 samples were lost; PCR protocols to determine the presence of an Arcobacter genus hence, broths from 70 samples were analyzed. PCR analysis amplicon (6). When the genus amplicon was detected in isolates, of these enrichment broths (n ϭ 420) showed that using species-specific amplicons (14) (Table 2) were then sought. The method B, 37% (26 of 70) of samples were positive for former protocol was modified as previously described (29) and was further modified to include a ‘‘universal’’ primer pair to act arcobacters, while method D yielded 36% (25 of 70), meth- as an internal control for each individual assay. The universal od A 13% (9 of 70), and method C 9% (6 of 70). The primers were used at a concentration of 2.5 ␮m for primer U1 ranking of these results is in agreement with those in Table and 1.25 ␮m for primer U2. An amplicon of 408 bp indicated 3, indicating that methods A and C are less effective for presence of bacterial DNA. The genus-specific amplicon was 331 recovery of arcobacters from red meats than are the other bp, while the species-specific amplicons were 401 bp (A. butzleri), two methods tested. 257 bp (A. cryaerophilus), and 641 bp (A. skirrowii). DISCUSSION Statistical analysis. Pairwise correlation analysis was con- ducted using GenStat Release 8.2 (Lawes Agricultural Trust, Ro- This study was a relative comparison of four protocols thamstead, UK). and constraints on resources meant that only one suspect colony per plate was selected for identification. Therefore, RESULTS the apparent efficiency of the methods would be underes- Retail packs of beef (n ϭ 80) were examined for the timated, in comparison with studies where several colonies presence of arcobacters, using four isolation procedures were selected. However, this should not affect the aim of (Table 1). In total, 720 plates of selective media were ex- the study, which was to determine which of the protocols amined, yielding 213 isolates, of which 24 (11.3%) were detected the highest number of retail beef samples contain- genotypically confirmed as Arcobacter spp. Method D gave ing Arcobacter spp. Since only one colony per positive the highest number of positive samples (15%; Table 3) and plate was identified, it would be expected that the number recovery for this method was significantly higher than for of confirmed Arcobacter isolates would be less than the any of the other three isolation procedures (P Ͻ 0.01). corresponding number of Arcobacter-positive enrichment A. butzleri was the dominant species isolated by all broths. Given that the majority (88.7%) of suspect colonies four methods, comprising 88% of all Arcobacter isolated selected were not Arcobacter spp., a considerable number of contaminants with colony morphologies similar to those of arcobacters must have been present. Other studies have TABLE 3. Comparison of the percentage of samples yielding Ar- noted that Arcobacter selective media often allow signifi- cobacter spp. from 80 packs of beef by the four methods studieda cant growth of contaminants (12, 28, 29). Selecting a larger number of colonies from each plate, as would occur in the Positive Method samples (%) Reference routine application of a single protocol, would increase the yield of Arcobacter spp. and so ensure that the number of A 1.3 Houf et al. (13) Arcobacter-positive samples would approach the number of B 6.3 Johnson and Murano (17, 18) as modified positive enrichment broths. by Scullion et al. (28) A previous comparison of methods A and B (29) C 2.5 Atabay and Corry (4) showed that both methods detected arcobacters in the same D 15.0 This study number of retail packs of poultry meat, but method B was a There was no significant difference between methods A, B, and selected as the protocol of choice on the basis of overall C(P Ͼ 0.05), but method D yielded significantly more positive high recovery and greatest diversity of Arcobacter species. samples than did the other three methods (P Ͻ 0.01). In the current study, the recoveries for these two methods J. Food Prot., Vol. 71, No. 4 ISOLATION OF ARCOBACTER FROM BEEF 853 were again not statistically significantly different (Table 3). tachment of Arcobacter butzleri, a new waterborne pathogen, to wa- Scullion et al. (29) also noted that sampling of the enrich- ter distribution pipe surfaces. J. Food Prot. 65:1240–1247. 3. Atabay, H. I., F. Aydin, K. Houf, M. Sahin, and P. Vandamme. 2003. ment broth at 24 h as well as 48 h increased the number The prevalence of Arcobacter spp. on chicken carcasses sold in retail of positive samples found by 36%, indicating that methods markets in Turkey, and identification of the isolates using SDS- for the isolation of arcobacters from foodstuffs were not yet PAGE. Int. J. Food Microbiol. 81:21–28. optimal. In the study reported here, 12 positive samples 4. Atabay, H. I., and J. Corry. 1998. Evaluation of a new Arcobacter were found using method D, of which three were positive enrichment medium and comparison with two media developed for enrichment of Campylobacter spp. Int. J. Food Microbiol. 41:53– only on the 24-h sampling and six only on the 48-h sam- 58. pling. Thus, the addition of 24-h sampling to method D 5. Atabay, H. I., and J. E. L. Corry. 1997. The prevalence of campylo- was again seen to increase recovery of arcobacters. bacters and arcobacters in broiler chickens. J. Appl. Microbiol. 83: In addition, in this study, incubating both the enrich- 619–626. ment culture broths and the selective media in a microaer- 6. Bastyns, K., D. Cartuyvels, S. Chapelle, P. Vandamme, H. Goossens, and R. De Wachter. 1995. A variable 23S rDNA region is a useful obic cabinet with free access to the atmosphere (method D) discriminating target for genus specific and species-specific PCR am- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/71/4/850/1682209/0362-028x-71_4_850.pdf by guest on 27 September 2021 yielded significantly more Arcobacter-positive samples plification in Arcobacter species. Syst. Appl. Microbiol. 18:353–356. than did method B alone (Table 3). With method B, the 7. Collins, C. H., P. M. Lyne, and J. M. Grange. 1995. Collins and enrichment medium is incubated a sealed bottle, with a lim- Lyne’s microbiological methods, 7th ed. Butterworth-Heinemann, ited headspace, in air, followed by incubation of the selec- London. 8. Collins, C. I., I. V. Wesley, and E. A. Murano. 1995. Detection of tive medium in air. Since methods B and D gave the same Arcobacter spp. in ground pork by modified plating methods. J. number of enrichment broths that yielded Arcobacter DNA, Food Prot. 59:448–452. the higher recovery using method D must be due either to 9. Dhamabutra, N., P. Kamol-Rathanakul, and K. Pirnthaweechai. 1992. better survival and growth of Arcobacter spp., or to better Isolation of from the canals of Bangkok metropoli- suppression of the contaminating flora. In either case, the tan area. J. Med. Assoc. Thail. 75:350–364. 10. Fernandez, H., S. Krause, and M. P. Villanueva. 2004. Arcobacter media of Johnson and Murano (17, 18) gave the highest butzleri, an emerging enteropathogen: communication of two cases recovery of arcobacters, and incubation of all media in a with chronic diarrhea. Braz. J. Microbiol. 35:216–218. microaerobic cabinet significantly improved recovery of ar- 11. Harrington, C. S., F. M. Thompson-Carter, and P. 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