Review of current methodologies to isolate and identify Campylobacter spp. from foods Gregory Gharst a , Omar A. Oyarzabal b, ⁎, Syeda K. Hussain c a U.S. Food and Drug Administration, Bedford Park, IL, USA b IEH Laboratories and Consulting Group, Lake Forest Park, WA, USA c Department of Biological Sciences, Alabama State University, Montgomery, AL, USA abstract article info Article history: Received 2 April 2013 Received in revised form 17 July 2013 Accepted 17 July 2013 Available online 27 July 2013 Keywords: Aerobic enrichment Broiler Campylobacter jejuni Campylobacter coli Chicken Isolation This article summarizes the most effective protocols to isolate Campylobacter spp. (mainly Campylobacter jejuni and Campylobacter coli) from food, primarily poultry products, and includes a summary of the current methods recommended by the Food and Drug Administration and the U.S. Department of Agriculture in the USA, and ISO in Europe. The recommended temperature for incubation of the samples throughout the isolation procedure is 42 °C. The enrichment of the samples for 48 h, which can be performed under aerobic conditions, is recommended to achieve a detectable number of Campylobacter cells. Bolton broth or buffered peptone water supplemented with cefoperazone and amphotericin B is commonly used enrichment broths. The transfer of the enriched samples to plate media using membrane filters helps to obtain pure Campylobacter colonies. Charcoal cefoperazone deoxycholate (CCDA) is the best choice among all plate media. There is no need to add oxygen quenching substances or blood to enrichment broth for the isolation of Campylobacter spp. However, the addition of blood to plate media aids in differential identification of presumptive colonies. Phase contrast microscopy and latex agglutination tests are confirmatory tests for presumptive Campylobacter isolates. The use of multiplex polymerase chain reaction (mPCR) assays is the simplest and most rapid method to identify isolates to the species level. mPCR assays, or other methods assessing DNA sequence variations, will probably become the confirmation procedure of choice in the future. Recent work with retail broiler meat has revealed that the rinsing of meat is more sensitive for the recovery of naturally contaminated retail broiler meat than current reference methods and requires less time for preparation and processing of the samples. This protocol could be coupled with DNA-based methods for a fast screening of positive samples. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Campylobacteriosis is a highly prevalent foodborne disease in industrialized nations. In the US, campylobacteriosis is the third most frequent bacterial foodborne disease, with 0.8 million estimated cases that represent 8% of the overall estimated foodborne disease illnesses (Scallan et al., 2011). Besides the typical diarrheal syndrome, Campylobacter infection has been linked to Guillain–Barré syndrome, which is an autoimmune-mediated disorder of the peripheral nervous system that results in acute demyelinating polyneuropathy (Kaldor and Speed, 1984; Speed et al., 1984). Although there are more than 20 Campylobacter spp., Campylobacter jejuni and Campylobacter coli are the two most important species associ- ated to human disease (Man, 2011). C. jejuni accounts for 80–90% of the infections reported in the US (Gilliss et al., 2013). In the USA, a mean of 28 outbreaks per year has been reported for years 2003–2008 (Taylor et al., 2013). The consumption of undercooked poultry is considered a major risk factor for sporadic infections (Friedman et al., 2004). However, the analysis of outbreak data in the last 10 years identifies raw milk as the main vehicle of transmission of campylobacteriosis, with 29% of the outbreaks associated with dairy products versus 11% associated with poultry and 5% associated with produce (Painter et al., 2013; Taylor et al., 2013). C. jejuni has a relatively low infectious dose based on experimental infection studies. Robinson (1981) reported that approximately 500 organisms were enough to produce infection after the consumption of a C. jejuni strain isolated from a raw milk outbreak in 1979 (Robinson et al., 1979). A previous study reported an infectious dose of 10 6 Campylobacter cells (Steele and McDermott, 1984), but the origin of the strain used in that study is unknown. The last report on experimental infections in humans was published in 1988, when disease was induced in volunteers who were given doses as low as 800 Campylobacter cells of a C. jejuni strain originated from a milk outbreak. These experiments corroborated that a low number of cells (less than 1000) can produce disease in humans (Black et al., 1988). It is accepted that all C. jejuni strains from all food sources are equally pathogenic, although none of Journal of Microbiological Methods 95 (2013) 84–92 ⁎ Corresponding author at: 15300 Bothell Way NE, Lake Forest Park, WA 98155, USA. Tel.: +1 206 522 5432; fax: +1 206 306 8883. E-mail addresses: oaoyarzabal@gmail.com, omar@iehinc.com (O.A. Oyarzabal). 0167-7012/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mimet.2013.07.014 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth