Campylobacter jejuni loss of culturability in aqueous microcosms and ability to resuscitate in a mouse model Wally Baffone a, * , Annalisa Casaroli a , Barbara Citterio a , Lucia Pierfelici a , Raffaella Campana a , Emanuela Vittoria a , Emilio Guaglianone b , Gianfranco Donelli b a Istituto di Scienze Tossicologiche, Igienistiche ed Ambientali, Universita ` di Urbino ‘‘Carlo Bo’’, Via S. Chiara 27, 61029 Urbino, Italy b Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanita ` , Roma, Italy Received 30 September 2003; received in revised form 30 May 2005; accepted 7 August 2005 Abstract Water is known as one of the main transmission routes of Campylobacter and contributes to increase the number of sporadic infections and outbreaks. Campylobacter jejuni persists in the environment, especially in water, in a viable but non-culturable (VBNC) form that is thought to be a possible cause of water-borne outbreaks. In this study, we evaluated the loss of culturability and viability of 9 C. jejuni strains of clinical origin and one ATCC reference strain when kept at 4 -C in artificial sea water (ASW). Culturability was measured as colony-forming units while viability was evaluated by CTC-DAPI double staining and the combined CTC-specific fluorescent antibody technique (CTC-FA). When cultured on Columbia Agar plates, strains exhibited different growth profiles which allowed to classify them into three different groups. Both techniques used to monitor the viability of the bacterial cells showed that C. jejuni strains survived in the VBNC form in the microcosms through a period lasting from 138 to 152 days. The recovery of C. jejuni VBNC forms to culturability, as evidenced by cell division, was obtained by passage in the mouse intestine. Our results indicate that C. jejuni VBNC cells were able to remain in this state for a few months and regain their culturability after in vivo passage depending on their lasting in the VBNC state, which affects the number of respiring bacteria. In fact, the resuscitation was achieved when the number of respiring bacteria became higher than 10 4 cell/ml. Therefore, a relatively high microbial titer of respiring bacteria in the VBNC state seems to be important for the resuscitation and subsequent intestinal colonisation. D 2005 Elsevier B.V. All rights reserved. Keywords: Campylobacter jejuni VBNC; Culturability; Viability; Resuscitation ability 1. Introduction Campylobacter jejuni , a microaerophilic Gram-negative bacterium, is a major cause of food-borne diseases in most developed countries (Alary and Nadeau, 1990; Griffith and Park, 1990; Shanker et al., 1990). The main sources of infection appear to be undercooked poultry meat, untreated water, raw milk and pets (Griffith and Park, 1990; Adak et al., 1995). In particular, water is also thought to be one of the most significant transmission routes of campylobacter, and inadequately treated drinking water con- tributes to a number of sporadic infections and outbreaks (Thomas et al., 1999). In fact, the occurrence of C. jejuni from aquatic habitat (water, sewage and marine environments) has been reported in several countries (Owen et al., 1995; Hernandez et al., 1996; Maugeri et al., 2004) and the isolation of this microorganism from marine environment (Obiri-Danso and Jones, 1999) underlines that this habitat, although unfavourable, may represent a significant bacterial reservoir. Epidemiological studies have revealed that the digestive tract of several breeding animals, poultry in particular, represents the normal habitat of C. jejuni (Shanker et al., 1990; Pearson and Healing, 1992; Mason et al., 1996). However, when released in the environment, the microorganism must face a series of stressful conditions, such as nutrient starvation, osmotic shock and temperature variation, forcing it to activate survival strategies which include entering the viable but non-culturable 0168-1605/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2005.08.015 * Corresponding author. Tel.: +39 722 303543; fax: +39 722 303541. E-mail address: baffone@uniurb.it (W. Baffone). International Journal of Food Microbiology 107 (2006) 83 – 91 www.elsevier.com/locate/ijfoodmicro