Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth Note Comparison of the XTT and resazurin assays for quantification of the metabolic activity of Staphylococcus aureus biofilm Beatriz Alonso a,b , Raquel Cruces a,b , Adrián Pérez c , Carlos Sánchez-Carrillo a , María Guembe a,b,⁎ a Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario Gregorio Marañón, Madrid, Spain b Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain c Biology Department, School of Biology, Universidad Autónoma de Madrid, Madrid, Spain ARTICLE INFO Keywords: S. aureus Biofilm Resazurin XTT Metabolic activity ABSTRACT We assessed whether resazurin was as efficient as XTT in the measurement of the metabolic activity of 209 clinical Staphylococcus aureus biofilm using an vitro model comparing the percentage of formazan and resorufin. The overall categorical agreement was 61.2% (r = 0.024), which means that resazurin can not substitute XTT. Staphylococcus aureus biofilms are microbial communities that ad- here to a surface and become embedded in an extracellular matrix (Tote et al., 2008). They play a key role in the development of persistent device-associated infections such as endocarditis and osteomyelitis and are therefore responsible for increasing rates of morbidity and mortality (Wagner and Hansch, 2016; Siala et al., 2016; Kong et al., 2016). Over the past few years, biofilm has been quantified using several in vitro models (Coenye and Nelis, 2010; Pettit et al., 2005). The easiest and most common method is a static model based on 96-well plates (Stepanovic et al., 2007). The model serves to quantify both biomass and metabolic activity using compounds such as crystal violet (CV), 2,3- bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H- tetrazolium hydroxide reduction (XTT), resazurin, fluorogenic Syto9, and fluorescein diacetate (FDA) (Vandecandelaere et al., 2016). How- ever, the most widely used systems are CV and XTT, which are tech- nologically advanced and user-friendly (Peeters et al., 2008). CV is a basic dye that binds to negatively charged molecules present on both the surface of bacteria and the extracellular matrix of biofilm (Peeters et al., 2008). It is used for quantification of biofilm biomass, as it stains both viable and non-viable cells and is easily measured by absorbance at 550 nm (Peeters et al., 2008; Xu et al., 2016). XTT is a tetrazolium salt that is reduced to water-soluble orange formazan by mitochondrial enzymes. Metabolic activity has been shown to be proportional to production of formazan by measuring absorbance at 492 nm (Gabrielson et al., 2002; Ginouves et al., 2014; Peeters et al., 2008; Xu et al., 2016). XTT is used for quantification of metabolic activity. Resazurin is a less toxic and cheaper compound. Its application is less time-consuming than XTT, and it is used to quantify metabolic activity by mitochondrial action, which reduces the compound to co- lored, fluorescent resorufin(Peeters et al., 2008). Resazurin was re- cently reported to have appropriately quantified metabolic activity in microorganisms such as Candida spp., Mycobacterium tuberculosis, and oral microbiota (Dalecki et al., 2016; Doll et al., 2016; Khot et al., 2008; Nam et al., 2016; Pina et al., 2016; Tawakoli et al., 2016; Vandal et al., 2015; Zarei Mahmoudabadi et al., 2014). However, data regarding its use for quantification, although becoming popular, compared with XTT of biofilm in gram-positive bacteria such as S. aureus are scarce. The only study comparing both assays was performed in 4 S. aureus strains Pettit et al., 2009). In addition, intra-species discordance has been re- ported (Peeters et al., 2008; Pettit et al., 2009). Therefore, the corre- lation between XTT and resazurin needs to be assessed in depth in the quantification of metabolic activity in bacteria. Our objective was to assess whether resazurin was as efficient as XTT in the measurement of the metabolic activity of the biofilm of 209 clinical S. aureus strains. Metabolic activity was assessed in 209 S. aureus strains isolated from blood cultures obtained at Hospital General Universitario Gregorio Marañón, Madrid, Spain. We used a 96-well plate–based model with a 24-hour-old biofilm, as described elsewhere (Alonso et al., 2016; Miller et al., 2010). Briefly, a loopful of fresh culture of strains was inoculated in 20 ml of TSB and incubated overnight. After three cycle of wash-resuspension with PBS, inoculum was adjusted to 0.5 McFarland. Then, 100 μl of the http://dx.doi.org/10.1016/j.mimet.2017.06.004 Received 21 March 2017; Received in revised form 2 June 2017; Accepted 2 June 2017 ⁎ Corresponding author at: Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario “Gregorio Marañón”, C/. Dr. Esquerdo, 46, 28007 Madrid, Spain. E-mail address: maria.guembe@iisgm.es (M. Guembe). Journal of Microbiological Methods 139 (2017) 135–137 Available online 03 June 2017 0167-7012/ © 2017 Elsevier B.V. All rights reserved. MARK