“Limits of Control” – Crucial Parameters for a Reliable Quantification of Viable Campylobacter by Real-Time PCR

Abstract

The unsuitability of the “CFU” parameter and the usefulness of cultivation-independent quantification of Campylobacter on chicken products, reflecting the actual risk for infection, is increasingly becoming obvious. Recently, real-time PCR methods in combination with the use of DNA intercalators, which block DNA amplification from dead bacteria, have seen wide application. However, much confusion exists in the correct interpretation of such assays. Campylobacter is confronted by oxidative and cold stress outside the intestine. Hence, damage caused by oxidative stress probably represents the most frequent natural death of Campylobacter on food products. Treatment of Campylobacter with peroxide led to complete loss of CFU and to significant entry of any tested DNA intercalator, indicating disruption of membrane integrity. When we transiently altered the metabolic state of Campylobacter by abolishing the proton-motive force or by inhibiting active efflux, CFU was constant but enhanced entry of ethidium bromide (EtBr) was observed. Consistently, ethidium monoazide (EMA) also entered viable Campylobacter, in particular when nutrients for bacterial energization were lacking (in PBS) or when the cells were less metabolically active (in stationary phase). In contrast, propidium iodide (PI) and propidium monoazide (PMA) were excluded from viable bacterial cells, irrespective of their metabolic state. As expected for a diffusion-limited process, the extent of signal reduction from dead cells depended on the temperature, incubation time and concentration of the dyes during staining, prior to crosslinking. Consistently, free protein and/or DNA present in varying amounts in the heterogeneous matrix lowered the concentration of the DNA dyes at the bacterial membrane and led to considerable variation of the residual signal from dead cells. In conclusion, we propose an improved approach, taking into account principles of method variability and recommend the implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) of Campylobacter by real-time PCR.

Enumeration of viable non-culturable Vibrio cholerae using propidium monoazide combined with quantitative PCR

“…PMA does not readily penetrate the membrane of living cells, and it mainly relies on free diffusion to reach the DNA of cells with compromised membranes (Nocker et al, 2006). For live cells with intact membranes, previous studies suggested that cell uptake might explain the reduction in DNA detection (Nkuipou-Kenfack et al, 2013;Kruger et al, 2014). These two different principles result in different efficiencies with these two types of cell.…”

Effect of exposure to stress conditions on propidium monoazide (PMA)-qPCR based Campylobacter enumeration in broiler carcass rinses

“…As such, the optimized quantity of PMA might be a critical limiting factor if the optimization is made using a pure culture. Kruger et al (2014) suggested that in broiler carcass rinse the composition of the matrix severely affected PMA dead signal reduction. According to van Frankenhuyzen et al (2011) the turbidity of the sample can also pose a problem if optimization doesn't occur first.…”

“…Since cell membrane integrity is essential for bacteria viability, the dyes that are used to block the DNA originating from dead cells and their application to molecular methods have a huge potential. According to Kruger et al (2014) detecting cells that own an intact membrane, which is non-permeable to the widely accepted live/dead discriminatory DNA dye, would be more suitable to reflect the potential risk for infection by food products, in particular when the bacteria are stressed and lose their ability to grow on plates. For Dinu and Bach (2013) a high correlation between the PMA-qPCR results and the total viable counts detected by the fluorescence microscopy of Escherichia coli could be established.…”

“…Suggestions for a more reliable use of PMA were proposed by Kruger et al (2014). In the previous study an implementation of process sample controls for reliable quantification of intact and potentially infectious units (IPIU) instead of colony-forming units (CFU) was made.…”

Critical review on biofilm methods

“…However, this approach can overestimate the number of viable cells due to the presence of free extracellular DNA (eDNA) (Klein et al, 2012) and DNA derived from dead cells. To avoid quantification of DNA not derived from living cells, samples can be treated with propidium monoazide (PMA) prior to DNA extraction (Alvarez et al, 2013;Kruger et al, 2014;Yasunaga et al, 2013). This molecule enters only membrane-compromised cells, intercalating between bases, and also interacts with eDNA (Nocker et al, 2007;Waring, 1965).…”