Evaluation of four protocols for the detection and isolation of thermophilic Campylobacter from different matrices

Campylobacter infections pose a serious public health problem; the incidence of campylobacteriosis has progressively increased in developed countries, and the pathogen is now considered the leading cause of bacterial gastroenteritis throughout the world (Humphrey et al. 2007; FAO⁄WHO, 2009). Thermophilic Campylobacter jejuni and Campylobacter coli are the most frequently isolated species in foodborne zoonoses in humans (EFSA Journal, 2011). Campylobacter can establish itself as a subclinical infection in humans, but frequently causes a range of clinical symptoms varying from self-limited, mild diarrhea to severe inflammatory bloody diarrhoea. Occasionally, acute or long-term and potentially serious complications occur such as septicaemia, irritable bowel syndrome, reactive arthritis or autoimmune neuropathies (Guillain-Barre´ and Miller Fisher Syndrome) (Godschalk et al. 2004; Leonard et al. 2004; Takahashi et al. 2005; Humphrey et al. 2007). Large outbreaks are uncommon, and the vast majority of human campylobacteriosis cases are sporadic; they most likely result from handling or consumption of raw or undercooked contaminated meat products. Other foodstuffs, untreated drinking water and milk have also been associated with the illness, but poultry products are considered the major source of infection (Pebody et al. 1997; Altekruse et al. 1999; Pires et al. 2010).

Bacteriological culture of Campylobacter spp. can be a challenge, owing to the fragility of these organisms. The use of a selective medium is recommended for the recovery from stool and faeces; for samples with low bacterial numbers, filtration or enrichment steps are typically added to improve recovery (Hu and Kuo 2011). Direct plating on selective agar media is common practice for Campylobacter isolation from several matrices (drinking water, environmental (dust) or intestinal samples), but an ideal single method for the entire range of samples requiring testing has not been developed (Baylis et al. 2000; Engberg et al. 2000; Musgrove et al. 2001; Commission Decision 2007 ⁄ 516 ⁄ EC). In 2006, the International Organization for Standardization (ISO) standard method for detection of Campylobacter spp. in food recommended enrichment using Bolton broth, followed by culture on selective modified charcoal cefoperazone desoxycholate agar (mCCDA) and one other alternative agar plate (ISO, 2006).

For our study, which covered various matrices, we compared the results of traditional culturing methods and a real-time quantitative PCR assay, in an attempt to combine optimal sensitivity with short isolation and confirmation time. We evaluated three different procedures for Campylobacter isolation: direct plating on selective media [mCCDA or Campyfood Agar (CFA)], four combinations of enrichment and plating media (Bolton or Preston enrichment, combined with mCCDA or CFA plates) and molecular detection by real-time PCR (qPCR). The evaluation was performed on naturally contaminated broiler faeces, neck skin and poultry meat samples.