Detection and Quantification of Viable but Non-culturable Campylobacter jejuni.

Ruiling Lv,Kaidi Wang,Donghong Liu,Jinsong Feng,Dustin D Heeney,Xiaonan Lu

doi:10.3389/fmicb.2019.02920

Ruiling Lv, Kaidi Wang + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2019.02920

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Abstract

Campylobacter can enter a viable but non-culturable (VBNC) state to evade various stresses, and this state is undetectable using traditional microbiological culturing techniques. These VBNC bacterial cells retain metabolism and demonstrate pathogenic potential due to their ability to resuscitate under favorable conditions. Rapid and accurate determination of VBNC Campylobacter is critical to further understand the induction and resuscitation of the dormancy state of this microbe in the agri-food system. Here, we integrated propidium monoazide (PMA) with real-time polymerase chain reaction (qPCR) targeting the rpoB gene to detect and quantify Campylobacter jejuni in the VBNC state. First, we optimized the concentration of PMA (20 μM) that could significantly inhibit the amplification of dead cells by qPCR with no significant interference on the amplification of viable cell DNA. PMA-qPCR was highly specific to C. jejuni with a limit of detection (LOD) of 2.43 log CFU/ml in pure bacterial culture. A standard curve for C. jejuni cell concentrations was established with the correlation coefficient of 0.9999 at the linear range of 3.43 to 8.43 log CFU/ml. Induction of C. jejuni into the VBNC state by osmotic stress (i.e., 7% NaCl) was rapid (<48 h) and effective (>10% population). The LOD of PMA-qPCR for VBNC C. jejuni exogenously applied to chicken breasts was 3.12 log CFU/g. In conclusion, PMA-qPCR is a rapid, specific, and sensitive method for the detection and quantification of VBNC C. jejuni in poultry products. This technique can give insight into the prevalence of VBNC Campylobacter in the environment and agri-food production system.

Highlights

Campylobacter is responsible for the most frequently reported foodborne gastrointestinal infection in the world (Silva et al, 2011)
Unfavorable environmental conditions, such as desiccation, aerobic stress, and starvation, can induce C. jejuni to enter the viable but non-culturable (VBNC) state and evade detection using the culture-based methods (Baffone et al, 2006). These VBNC cells can remain viable for an extended time period, resuscitate under favorable conditions, recover metabolism and virulence (Ramamurthy et al, 2014; Pinto et al, 2015), and subsequently pose an important concern to the public health
Since poultry is one of the major sources of C. jejuni contamination and a common vehicle for foodborne transmission of Campylobacteriosis (Young et al, 2007), we further evaluated the performance of propidium monoazide (PMA)-quantitative PCR (qPCR) to quantify the model contamination of chicken meat with VBNC C. jejuni cells

Summary

Introduction

Campylobacter is responsible for the most frequently reported foodborne gastrointestinal infection in the world (Silva et al, 2011). 85 bacterial species have been described to form VBNC cells under stress conditions, including Escherichia, Vibrio, Listeria, and Campylobacter (Pinto et al, 2015). VBNC C. jejuni cells have been reported to resuscitate in vivo with mouse infections (Baffone et al, 2006), in microaerobic conditions (Bovill and Mackey, 1997), and in embryonated chicken eggs (Cappelier et al, 1999). VBNC cells that become resuscitated can regain full infective phenotypes (Baffone et al, 2006; Pinto et al, 2015), representing a real threat to the public health

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