Skip to main content

Advertisement

SpringerLink
Log in

Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment

  • Methods and protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 μM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Agustí G, Codony F, Fittipaldi M, Adrados B, Morato J (2010) Viability determination of Helicobacter pylori using propidium monoazide quantitative PCR. Helicobacter 15:473–476

    Article  Google Scholar 

  • Andorrà I, Esteve-Zarzoso B, Guillamón JM, Mas A (2010) Determination of viable wine yeast using DNA binding dyes and quantitative PCR. Int J Food Microbiol 144:257–262

    Article  Google Scholar 

  • Brescia CC, Griffin SM, Ware MW, Varughese EA, Egorov AI, Villegas EN (2009) Cryptosporidium propidium monoazide-PCR, a molecular biology-based technique for genotyping of viable Cryptosporidium oocysts. Appl Environ Microbiol 75:6856–6863

    Article  CAS  Google Scholar 

  • Brion LP, Uko SE, Goldman DL (2007) Risk of resistance associated with fluconazole prophylaxis: systematic review. J Infect 54:521–529

    Article  Google Scholar 

  • Calugi C, Trabocchi A, Guarna A (2011) Novel small molecules for the treatment of infections caused by Candida albicans: a patent review (2002–2010). Expert Opin Ther Patents 21:381–397

    Article  CAS  Google Scholar 

  • Cawthorn DM, Witthuhn RC (2008) Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromidemonoazide. J Appl Microbiol 105:1178–1185

    Article  CAS  Google Scholar 

  • Chang B, Sugiyama K, Taguri T, Amemura-Maekawa J, Kura F, Watanabe H (2009) Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/Real-Time PCR. Appl Environ Microbiol 75:147–153

    Article  CAS  Google Scholar 

  • Chang B, Taguri T, Sugiyama K, Amemura-Maekawa J, Kura F, Watanabe H (2010) Comparison of ethidium monoazide and propidium monoazide for the selective detection of viable Legionella cells. Jpn J Infect Dis 63:119–123

    CAS  Google Scholar 

  • Elizaquível P, Sánchez G, Selma MV, Aznar R (2012) Application of propidium monoazide-qPCR to evaluate the ultrasonic inactivation of Escherichia coli O157:H7 in fresh-cut vegetable wash water. Food Microbiol 30:316–320

    Article  Google Scholar 

  • Fittipaldi M, Rodriguez NJP, Codony F, Adrados B, Peñuela GA, Morató J (2010) Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR. J Virol Methods 168:228–232

    Article  CAS  Google Scholar 

  • Fittipaldi M, Rodriguez NJP, Adrados B, Agustí G, Peñuela G, Morató J, Codony F (2011) Discrimination of viable Acanthamoeba castellani trophozoites and cysts by propidium monoazide real-time polymerase chain reaction. J Eukaryot Microbiol 58:359–364

    Article  Google Scholar 

  • 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. doi:10.1016/j.mimet.2012.08.007

  • Flekna G, Stefanic P, Wagner M, Smulders FJ, Mozina SS, Hein I (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

    Article  CAS  Google Scholar 

  • Fricke S, Fricke C, Schimmelpfennig C, Oelkrug C, Schönfelder U, Blatz R, Zilch C, Faber S, Hilger N, Ruhnke M, Rodloff AC (2010) A real-time PCR assay for the differentiation of Candida species. J Appl Microbiol 109:1150–1148

    Article  CAS  Google Scholar 

  • Garnacho-Montero J, Díaz-Martín A, García-Cabrera E, Pérez R, de Pipaón M, Hernández-Caballero C, Aznar-Martín J (2010) Risk factors for fluconazole-resistant candidemia. Antimicrob Agents Chemother 54:3149–3154

    Article  CAS  Google Scholar 

  • Graybill JR (2000) Systemic antifungal drugs. In: Kushwaha RKS, Guarro J (ed) Bilbao (Spain). Biology of dermatophytes and other keratinophyilic fungi. Revista Iberoamericana de Micologia, pp 168–174

  • Gurib-Fakin A (2006) Medicinal plants: traditions of yesterday and drugs of tomorrow. Mol Aspects Med 27:01–93

    Article  Google Scholar 

  • Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Biotechnology 11:1026–1030

    Google Scholar 

  • Josephson KL, Gerba CP, Pepper IL (1993) Polymerase chain reaction detection of nonviable bacterial pathogens. Appl Environ Microbiol 59:3513–3515

    CAS  Google Scholar 

  • Khan Z, Mustafa AS, Alam FF (2009) Real-time LightCycler polymerase chain reaction and melting temperature analysis for identification of clinically important Candida spp. J Microbiol Immunol Infect 42:290–295

    CAS  Google Scholar 

  • Kollef M, Micek S, Hampton N, Doherty JA, Kumar A (2012) Septic shock attributed to Candida infection: importance of empiric therapy and source control. Clin Infect Dis 54:1739–1746

    Article  CAS  Google Scholar 

  • 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:S80–S86

    Article  CAS  Google Scholar 

  • Kramer M, Obermajer N, Matijašić BB, Rogelj I, Kmetec V (2009) Quantification of live and dead probiotic bacteria in lyophilised product by real-time PCR and by flow cytometry. Appl Microbiol Biotechnol 84:1137–1147

    Article  CAS  Google Scholar 

  • Maaroufi Y, Heymans C, De Bruyne JM, Duchateau V, Rodriguez-Villalobos H, Aoun M, Crokaert F (2003) Rapid detection of Candida albicans in clinical blood samples by using a TaqMan-based PCR assay. J Clin Microbiol 41:3293–3298

    Article  CAS  Google Scholar 

  • Marr KA (2009) Fungal infections in oncology patients: update of epidemiology, prevention, and treatment. Curr Opin Oncol 22:138–142

    Article  Google Scholar 

  • Martínez M, López-Ribot JL, Kirkpatrick WR, Bachmann SP, Perea S, Ruesga MT, Patterson TF (2002) Heterogeneous mechanisms of azole resistance in Candida albicans clinical isolates from an HIV-infected patient on continuous fluconazole therapy for oropharyngeal candidosis. J Antimicrob Chemother 49:515–524

    Article  Google Scholar 

  • Miceli MH, Díaz JA, Lee SA (2011) Emerging opportunistic yeast infections. Lancet Infect Dis 11:142–151

    Article  Google Scholar 

  • Minami J, Yoshida K, Soejima T, Yaeshima T, Iwatsuki K (2010) New approach to use ethidium bromide monoazide as an analytical tool. J Appl Microbiol 109:900–909

    Article  CAS  Google Scholar 

  • Miotto P, Bigoni S, Migliori GB, Matteelli A, Cirillo DM (2012) Early tuberculosis treatment monitoring by Xpert® MTB/RIF. Eur Respir J 39:1269–1271

    Article  Google Scholar 

  • Nam S, Kwon S, Kim MJ, Chae JC, Jae Maeng P, Park JG, Lee GC (2011) Selective detection of viable Helicobacter pylori using ethidium monoazide or propidium monoazide in combination with real-time polymerase chain reaction. Microbiol Immunol 55:841–846

    Article  CAS  Google Scholar 

  • Nocker A, Camper AK (2006) Selective removal of DNA from dead cells of mixed bacterial communities by use of ethidium monoazide. Appl Environ Microbiol 72:1997–2004

    Article  CAS  Google Scholar 

  • Nocker A, Camper AK (2009) Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques. FEMS Microbiol Lett 291:137–142

    Article  CAS  Google Scholar 

  • Nocker A, Cheung CY, Camper AK (2006) Comparison of propidium monoazide and ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells. J Microbiol Methods 67:310–320

    Article  CAS  Google Scholar 

  • Nogva HK, Drømtorp SM, Nissen H, Rudi K (2003) Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5′-nuclease PCR. Biotechniques 34:804–813

    CAS  Google Scholar 

  • Pan Y, Breidt F (2007) Enumeration of viable Listeria monocytogenes cells by real-time PCR with propidium monoazide and ethidium monoazide in the presence of dead cells. Appl Environ Microbiol 73:8028–8031

    Article  CAS  Google Scholar 

  • Pappas PG, Rex JH, Sobel JD, Filler SG, Dismukes WE, Thomas J, Walsh TJ, Edwards JE (2004) Guidelines for treatment of candidiasis. Clin Infect Dis 38:161–189

    Article  Google Scholar 

  • Parshionikar S, Laseke I, Fout GS (2010) Use of propidium monoazide in reverse transcriptase polymerase chain reactions to distinguish between infectious and non-infectious enteric viruses in water. Appl Environ Microbiol 76:4318–4326

    Article  CAS  Google Scholar 

  • Pfaller MA, Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20:133–163

    Article  CAS  Google Scholar 

  • Rasooli I, Owlia P (2005) Chemoprevention by thyme oils of Aspergillus parasiticus growth and aflatoxin production. Phytochemistry 66:2851–2856

    Article  CAS  Google Scholar 

  • Rawsthorne H, Phister TG (2009) Detection of viable Zygosaccharomyces bailii in fruit juices using ethidium monoazide bromide and real-time PCR. Int J Food Microbiol 131:246–250

    Article  CAS  Google Scholar 

  • Rawsthorne H, Dock CN, Jaykus LA (2009) PCR-based method using propidium monoazide to distinguish viable from nonviable Bacillus subtilis spores. Appl Environ Microbiol 75:2936–2939

    Article  CAS  Google Scholar 

  • Rudi K, Moen B, Drømtorp 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

    Article  CAS  Google Scholar 

  • Sánchez G, Elizaquível P, Aznar R (2012) Discrimination of infectious hepatitis A viruses by propidium monoazide real-time RT-PCR. Food Environ Virol 4:21–25

    Article  Google Scholar 

  • Shi H, Xu W, Trinh Q, Luo Y, Liang Z, Li Y, Huang K (2012) Establishment of a viable cell detection system for microorganisms in wine based on ethidium monoazide and quantitative PCR. Food Control 27:81–86

    Article  Google Scholar 

  • Shin JH, Nolte FS, Holloway BP, Morrison CJ (1999) Rapid identification of up to three Candida species in a single reaction tube by a 59 exonuclease assay using fluorescent DNA probes. J Clin Microbiol 37:165–170

    CAS  Google Scholar 

  • Sims CR, Ostrosky-Zeichner L, Rex JH (2005) Invasive candidiasis in immunocompromised hospitalized patients. Arch Med Res 36:660–671

    Article  Google Scholar 

  • Takahashi Y, Yoshida A, Nagayoshi N, Kitamura C, Nishihara T, Awano S, Ansai T (2011) Enumeration of viable Enterococcus faecalis, a predominant apical periodontitis pathogen, using propidium monoazide and quantitative real-time polymerase chain reaction. Microbiol Immunol 55:889–892

    Article  CAS  Google Scholar 

  • Thomas MC, Selinger LB, Douglas G (2012) Seasonal diversity of planktonic protists in southwestern Alberta Rivers over a one year period revealed by T-RFLP and 18S rRNA gene libraries. Appl Environ Microbiol. doi:10.1128/AEM.00237-12

  • Tsai ΜL, Lin CC, Lin WC, Yang CH (2011) Antimicrobial, antioxidant, and anti-inflammatory activities of essential oils from five selected herbs. Biosci Biotechnol Biochem 75:1977–1983

    Article  CAS  Google Scholar 

  • Turenne CY, Sanchez SE, Hoban DJ, Karlowsky JA, Kabani AM (1999) Rapid identification of fungi by using the ITS2 genetic region and an automated fluorescent capillary electrophoresis system. J Clin Microbiol 37:1846–1851

    CAS  Google Scholar 

  • van de Vossenberg JL, Ubbink-kok T, Elferink MG, Driessen AJ, Konings WN (1995) Ion permeability of the cytoplasmatic membrane limits the maximum growth temperature of bacteria and archaea. Mol Microbiol 18:925–932

    Article  Google Scholar 

  • van Frankenhuyzen JK, Trevors JT, Lee H, Flemming CA, Habash MB (2011) Molecular pathogen detection in biosolids with a focus on quantitative PCR using propidium monoazide for viable cell enumeration. J Microbiol Methods 87:263–272

    Article  Google Scholar 

  • van Vuuren SF, Suliman S, Viljoen AM (2009) The antimicrobial activity of four commercial essential oils in combination with conventional antimicrobials. Lett Appl Microbiol 48:440–446

    Article  Google Scholar 

  • Vesper S, McKinstry C, Hartmann C, Neace M, Yoder S, Vesper A (2008) Quantifying fungal viability in air and water samples using quantitative PCR after treatment with propidium monoazide (PMA). J Microbiol Methods 72:180–184

    Article  CAS  Google Scholar 

  • Wagner AO, Malin C, Knapp BA, Illmer P (2008) Removal of free extracellular DNA from environmental samples by ethidium monoazide and propidium monoazide. Appl Environ Microbiol 74:2537–2539

    Article  CAS  Google Scholar 

  • Wang L, Li Y, Mustapha A (2009) Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR. J Appl Microbiol 107:1719–1728

    Article  CAS  Google Scholar 

  • Yañez MA, Nocker A, Soria-Soria E, Múrtula R, Martinez L, Catalán V (2011) Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR. J Microbiol Methods 85:124–130

    Article  Google Scholar 

Download references

Acknowledgments

This research was supported by a grant (“Convocatòria d’Ajuts per a la Iniciació i Reincorporació a la Recerca”) awarded to Gemma Agustí from Polytechnic University of Catalonia.

Author information

Authors and Affiliations

  1. Laboratori de Microbiologia Sanitaria i Mediambiental (MSM-Lab) & Aquasost-UNESCO Chair in Sustainability, Universitat Politècnica de Catalunya (UPC), Edifici Gaia, Pg. Ernest Lluch/Rambla Sant Nebridi, 08222, Terrassa, Barcelona, Spain

    Gemma Agustí, Mariana Fittipaldi, Jordi Morató & Francesc Codony

Authors
  1. Gemma Agustí
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mariana Fittipaldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jordi Morató
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Francesc Codony
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Francesc Codony.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Agustí, G., Fittipaldi, M., Morató, J. et al. Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment. Appl Microbiol Biotechnol 97, 341–349 (2013). https://doi.org/10.1007/s00253-012-4524-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4524-z

Keywords

Search

Navigation