Abstract
The genus Arcobacter (Vandamme et al., 1991), comprised of Campylobacter-related species, are considered zoonotic emergent pathogens. The presence of Arcobacter in food products like shellfish, has an elevated incidence worldwide. In this study, we developed a specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The high selectivity of primers was demonstrated by using purified DNA from 38 different species, 20 of them from the genus Arcobacter. The optimization of PMA concentration showed that 20 μM was considered as an optimal concentration that inhibits the signal from dead cells at different concentrations (OD550 from 0.2 to 0.8) and at different ratios of live: dead cells (50:50 and 90:10). The v-qPCR results from shellfish samples were compared with those obtained in parallel using several culture isolation approaches (i.e., direct plating on marine and blood agar and by post-enrichment culturing in both media). The enrichment was performed in parallel in Arcobacter-CAT broth with and without adding NaCl. Additionally, the v-qPCR results were compared to those obtained with traditional quantitative (qPCR). The v-qPCR and the qPCR resulted in c.a. 94% of positive detection of Arcobacter vs. 41% obtained by culture approaches. When examining the reduction effect resulting from the use of v-qPCR, samples pre-enriched in Arcobacter-CAT broth supplemented with 2.5% NaCl showed a higher reduction (3.27 log copies) than that of samples obtained directly and those pre-enriched in Arcobacter-CAT broth isolation (1.05 and 1.04). When the v-qPCR was applied to detect arcobacter from real shellfish samples, 15/17 samples tested positive for viable Arcobacter with 3.41 to 8.70 log copies 1g-1. This study offers a new tool for Arcobacter surveillance in seafood.
Highlights
Foodborne disease outbreaks are of public health concern (Zeng et al, 2016 and references therein)
Strains were grown on tryptone soy agar at 37◦C for 24 h (TSA, Difco, France), with the exception of Arcobacter species that were grown on Blood Agar (BA; TSA supplemented with 5% sheep blood BD Difco, Le Pont de Claix, France), and A. marinus and A. halophilus, that were grown on Marine Agar (MA; Scharlab, Barcelona, Spain) and were incubated under aerobic conditions at 30◦C for 48 h
Our results have shown that implementation of viable quantitative Polymerase Chain Reaction (PCR) (v-Quantitative PCR (qPCR)), using propidium monoazide (PMA), reduces the signal obtained from samples containing dead cells as compared to those analyzed by standard qPCR, indicating that amplification of free DNA, or that of dead cells, is being blocked as occurred for other bacterial species in many different samples (Kobayashi et al, 2009; Josefsen et al, 2010; Li and Chen, 2012; Zhu et al, 2012; Zhang et al, 2015)
Summary
Foodborne disease outbreaks are of public health concern (Zeng et al, 2016 and references therein). In 2015, a total of 4,362 foodborne disease outbreaks, including waterborne disease outbreaks were reported in the European Union (EU) Overall, these outbreaks caused 45,874 cases of illness, 3,892 hospitalizations and 17 deaths (EFSA and ECDC, 2016). To avoid the occurrence of disease outbreaks, food is monitored following specific microbiological criteria, which may vary according to culture, climate and economic status of the country (Zhang et al, 2016). In these regulated monitoring programs, the most commonly used methods are based on bacterial isolation in synthetic media, which are time consuming, laborious and cannot detect viable-but-non-culturable bacteria (VBNC) (Barbau-Piednoir et al, 2014)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
