Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.

Department of Experimental Biology Faculty of Science Masaryk University Brno Czechia

Department of Microbiology and Antimicrobial Resistance Veterinary Research Institute Brno Czechia

Laboratory of Neurobiology and Pathological Physiology Institute of Animal Physiology and Genetics Czech Academy of Sciences Libechov Czechia

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Appleton T. G., Hall J. R., Williams M. A. (1982). Amino-acid complexes of platinum(IV). VI. Ethylenediamine-tetraacetate (EDTA) complexes. Inorgan. Chim. Acta Article. 61 51–56.

Burden D. (2012). Guide to the disruption of biological samples - 2012. Random Prim. 25 1–25.

Busch D. H., Bailar J. C. (1956). The stereochemistry of complex inorganic compounds. XX. The tetradentate and bidentate complexes of ethylenediaminetetraacetic acid. J. Am. Chem. Soc. 78 716–719. 10.1021/ja01585a007 DOI

Canh V. D., Kasuga I., Furumai H., Katayama H. (2019). Viability RT-qPCR combined with sodium deoxycholate pre-treatment for selective quantification of infectious viruses in drinking water samples. Food Environ. Virol. 11 40–51. 10.1007/s12560-019-09368-2 PubMed DOI

Elguezabal N., Bastida F., Sevilla I. A., Gonzalez N., Molina E., Garrido J. M., et al. (2011). Estimation of Mycobacterium avium subsp. paratuberculosis growth parameters: strain characterization and comparison of methods. Appl. Environ. Microbiol. 77 8615–8624. 10.1128/Aem.05818-11 PubMed DOI PMC

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. 10.1016/j.mimet.2012.08.007 PubMed DOI

Fraisse A., Niveau F., Hennechart-Collette C., Coudray-Meunier C., Martin-Latil S., Perelle S. (2018). Discrimination of infectious and heat-treated norovirus by combining platinum compounds and real-time RT-PCR. Int. J. Food Microbiol. 269 64–74. 10.1016/j.ijfoodmicro.2018.01.015 PubMed DOI

Hall M. D., Telma K. A., Chang K. E., Lee T. D., Madigan J. P., Lloyd J. R., et al. (2014). Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes. Cancer Res. 74 3913–3922. 10.1158/0008-5472.Can-14-0247 PubMed DOI PMC

Kralik P., Beran V., Pavlik I. (2012). Enumeration of Mycobacterium avium subsp. paratuberculosis by quantitative real-time PCR, culture on solid media and optical densitometry. BMC Res. Notes 5:114. 10.1186/1756-0500-5-114 PubMed DOI PMC

Kralik P., Nocker A., Pavlik I. (2010). Mycobacterium avium subsp. paratuberculosis viability determination using F57 quantitative PCR in combination with propidium monoazide treatment. Int. J. Food Microbiol. 141 80–86. 10.1016/j.ijfoodmicro.2010.03.018 PubMed DOI

Nocker A., Camper A. K. (2009). Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. Fems Microbiol. Lett. 291 137–142. 10.1111/j.1574-6968.2008.01429.x PubMed DOI

Nocker A., Cheung C. Y., Camper A. K. (2006). Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J. Microbiol. Methods. 67 310–320. 10.1016/j.mimet.2006.04.015 PubMed DOI

Nocker A., Sossa K. E., Camper A. K. (2007). Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. J. Microbiol. Methods 70 252–260. 10.1016/j.mimet.2007.04.014 PubMed DOI

Nogva H. K., Dromtorp S. M., Nissen H., Rudi K. (2003). Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5 ‘-nuclease PCR. Biotechniques 34 804–813. 10.2144/03344rr02 PubMed DOI

Pickup R. W., Rhodes G., Arnott S., Sidi-Boumedine K., Bull T. J., Weightman A., et al. (2005). Mycobacterium avium subsp. paratuberculosis in the catchment area and water of the river Taff in South Wales, United Kingdom, an its potential relationship to clustering of Crohn’s disease cases in the city of Cardiff. Appl. Environ. Microbiol. 71 2130–2139. 10.1128/Aem.71.4.2130-2139.2005 PubMed DOI PMC

Puente H., Randazzo W., Falco I., Carvajal A., Sanchez G. (2020). Rapid selective detection of potentially infectious porcine epidemic diarrhea coronavirus exposed to heat treatments using viability RT-qPCR. Front. Microbiol. 11:1911. 10.3389/fmicb.2020.01911 PubMed DOI PMC

Randazzo W., Vasquez-Garcia A., Aznar R., Sanchez G. (2018). Viability RT-qPCR to distinguish between HEV and HAV with intact and altered capsids. Front. Microbiol. 9:1973. 10.3389/fmicb.2018.01973 PubMed DOI PMC

Sevilla I. A., Molina E., Elguezabal N., Perez V., Garrido J. M., Justea R. A. (2015). Detection of mycobacteria, Mycobacterium avium subspecies, and Mycobacterium tuberculosis complex by a novel tetraplex real-time PCR assay. J. Clin. Microbiol. 53 930–940. 10.1128/Jcm.03168-14 PubMed DOI PMC

Shi H., Xu W., Luo Y., Chen L., Liang Z., Zhou X., et al. (2011). The effect of various environmental factors on the ethidium monazite and quantitative PCR method to detect viable bacteria. J. Appl. Microbiol. 111 1194–1204. 10.1111/j.1365-2672.2011.05125.x PubMed DOI

Slana I., Kralik P., Kralova A., Pavlik I. (2008). On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int. J. Food Microbiol. 128 250–257. 10.1016/j.ijfoodmicro.2008.08.013 PubMed DOI

Soejima T., Iwatsuki K. (2016). Innovative use of palladium compounds to selectively detect live Enterobacteriaceae in milk by PCR. Appl. Environ. Microbiol. 82 6930–6941. 10.1128/Aem.01613-16 PubMed DOI PMC

Soejima T., Minami J., Xiao J., Abe F. (2016). Innovative use of platinum compounds to selectively detect live microorganisms by polymerase chain reaction. Biotechnol. Bioeng. 113 301–310. 10.1002/bit.25711 PubMed DOI

Yi Y. W., Bae I. (2011). Effects of solvents on in vitro potencies of platinum compounds. DNA Repair 10 1084–1085. 10.1016/j.dnarep.2011.09.008 PubMed DOI PMC

A viability assay combining palladium compound treatment with quantitative PCR to detect viable Mycobacterium avium subsp. paratuberculosis cells