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.
References
[1]
El-Shibiny A, Connerton P, Connerton I (2009) Survival at refrigeration and freezing temperatures of Campylobacter coli and Campylobacter jejuni on chicken skin applied as axenic and mixed inoculums. Int J Food Microbiol 131: 197–202. doi: 10.1016/j.ijfoodmicro.2009.02.024
[2]
Stingl K, Knüver M-T, Vogt P, Buhler C, Krüger N-J, et al (2012) Quo vadis? - Monitoring Campylobacter in Germany. Eur J Microbiol Immunol 2: 88–96. doi: 10.1556/eujmi.2.2012.1.12
[3]
EFSA (2011) Scientific Opinion on Campylobacter in broiler meat production: control options and performance objectives and/or targets at different stages of the food chain. EFSA Journal 9: 2105.
[4]
Bolton PH, Kearns DR (1978) Spectroscopic properties of ethidium monoazide: a fluorescent photoaffinity label for nucleic acids. Nucleic Acids Res 5: 4891–4903. doi: 10.1093/nar/5.12.4891
[5]
Nogva HK, Dromtorp SM, Nissen H, Rudi K (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. Biotechniques 34: 804–3.
[6]
Rudi K, Moen B, Dromtorp SM, Holck AL (2005) Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Appl Environ Microbiol 71: 1018–1024. doi: 10.1128/aem.71.2.1018-1024.2005
[7]
Josefsen MH, Lofstrom C, Hansen TB, Christensen LS, Olsen JE, et al (2010) Rapid quantification of viable Campylobacter bacteria on chicken carcasses, using real-time PCR and propidium monoazide treatment, as a tool for quantitative risk assessment. Appl Environ Microbiol 76: 5097–5104. doi: 10.1128/aem.00411-10
[8]
Pacholewicz E, Swart A, Lipman LJ, Wagenaar JA, Havelaar AH, et al. (2013) Propidium monoazide does not fully inhibit the detection of dead Campylobacter on broiler chicken carcasses by qPCR. J Microbiol Methods 95: 32–38. doi: 10.1016/j.mimet.2013.06.003
[9]
Fittipaldi M, Nocker A, Codony F (2012) Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification. J Microbiol Methods 91: 276–289. doi: 10.1016/j.mimet.2012.08.007
[10]
Rossmanith P, Roder B, Fruhwirth K, Vogl C, Wagner M (2011) Mechanisms of degradation of DNA standards for calibration function during storage. Appl Microbiol Biotechnol 89: 407–417. doi: 10.1007/s00253-010-2943-2
[11]
Lübeck PS, Wolffs P, On SL, Ahrens P, Radstrom P, et al. (2003) Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation. Appl Environ Microbiol 69: 5664–5669. doi: 10.1128/aem.69.9.5664-5669.2003
[12]
Josefsen MH, Jacobsen NR, Hoorfar J (2004) Enrichment followed by quantitative PCR both for rapid detection and as a tool for quantitative risk assessment of food-borne thermotolerant campylobacters. Appl Environ Microbiol 70: 3588–3592. doi: 10.1128/aem.70.6.3588-3592.2004
[13]
Anderson A, Pietsch K, Zucker R, Mayr A, Müller-Hohe E, et al. (2011) Validation of a duplex real-time PCR for the detection of Salmonella spp. in different food products. Food Anal Methods 4: 259–267. doi: 10.1007/s12161-010-9142-8
[14]
van Alphen LB, Burt SA, Veenendaal AK, Bleumink-Pluym NM, van Putten JP (2012) The natural antimicrobial carvacrol inhibits Campylobacter jejuni motility and infection of epithelial cells. PLoS One 7: e45343. doi: 10.1371/journal.pone.0045343
[15]
Mamelli L, Prouzet-Mauleon V, Pages JM, Megraud F, Bolla JM (2005) Molecular basis of macrolide resistance in Campylobacter: role of efflux pumps and target mutations. J Antimicrob Chemother 56: 491–497. doi: 10.1093/jac/dki253
[16]
Nebe-von-Caron G, Stephens PJ, Hewitt CJ, Powell JR, Badley RA (2000) Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J Microbiol Methods 42: 97–114. doi: 10.1016/s0167-7012(00)00181-0
Flekna G, Stefanic P, Wagner M, Smulders FJ, Mozina SS, et al. (2007) Insufficient differentiation of live and dead Campylobacter jejuni and Listeria monocytogenes cells by ethidium monoazide (EMA) compromises EMA/real-time PCR. Res Microbiol 158: 405–412. doi: 10.1016/j.resmic.2007.02.008
[19]
Kobayashi H, Oethinger M, Tuohy MJ, Hall GS, Bauer TW (2009) Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide. Lett Appl Microbiol 48: 633–638. doi: 10.1111/j.1472-765x.2009.02585.x
[20]
Loozen G, Boon N, Pauwels M, Quirynen M, Teughels W (2011) Live/dead real-time polymerase chain reaction to assess new therapies against dental plaque-related pathologies. Mol Oral Microbiol 26: 253–261. doi: 10.1111/j.2041-1014.2011.00615.x
[21]
Chang B, Taguri T, Sugiyama K, Amemura-Maekawa J, Kura F, et al. (2010) Comparison of ethidium monoazide and propidium monoazide for the selective detection of viable Legionella cells. Jpn J Infect Dis 63: 119–123. doi: 10.1128/aem.00604-08
[22]
Nkuipou-Kenfack E, Engel H, Fakih S, Nocker A (2013) Improving efficiency of viability-PCR for selective detection of live cells. J Microbiol Methods 93: 20–24. doi: 10.1016/j.mimet.2013.01.018
[23]
Rossmanith P, Mester P, Fruhwirth K, Fuchs S, Wagner M (2011) Proof of concept for recombinant cellular controls in quantitative molecular pathogen detection. Appl Environ Microbiol 77: 2531–2533. doi: 10.1128/aem.02601-10