MLPA diagnostics of complex microbial communities: Relative quantification of bacterial species in oral biofilms Zewdu Terefework a,b,c, ⁎, Chi L. Pham a,b , Anja C. Prosperi a,b , Mark M. Entius c , Abdellatif Errami c , Rob J.M. van Spanning a,b , Egija Zaura a , Jacob M. ten Cate a , Wim Crielaard a a Department of Cariology, Endodontology, Pedodontology, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, The Netherlands b Department of Molecular Cell Physiology, VU University Amsterdam, The Netherlands c MRC-Holland BV, Amsterdam, The Netherlands abstract article info Article history: Received 10 June 2008 Received in revised form 1 August 2008 Accepted 29 August 2008 Available online 13 September 2008 Keywords: Oral biofilms MLPA DGGE CDFF A multitude of molecular methods are currently used for identification and characterization of oral biofilms or for community profiling. However, multiplex PCR techniques that are able to routinely identify several species in a single assay are not available. Multiplex Ligation-dependent Probe Amplification (MLPA) identifies up to 45 unique fragments in a single tube PCR. Here we report a novel use of MLPA in the relative quantification of targeted microorganisms in a community of oral microbiota. We designed 9 species specific probes for: Actinomyces gerencseriae, Actinomyces naeslundii, Actinomyces odontolyticus, Candida albicans, Lactobacillus acidophilus, Rothia dentocariosa, Streptococcus mutans, Streptococcus sanguinis and Veillonella parvula; and genus specific probes for selected oral Streptococci and Lactobacilli based on their 16S rDNA sequences. MLPA analysis of DNA pooled from the strains showed the expected specific MLPA products. Relative quantification of a serial dilution of equimolar DNA showed that as little as 10 pg templates can be detected with clearly discernible signals. Moreover, a 2 to 7% divergence in relative signal ratio of amplified probes observed from normalized peak area values suggests MLPA can be a cheaper alternative to using qPCR for quantification. We observed 2 to 6 fold fluctuations in signal intensities of MLPA products in DNAs isolated from multispecies biofilms grown in various media for various culture times. Furthermore, MLPA analyses of DNA isolated from saliva obtained from different donors gave a varying number and intensity of signals. This clearly shows the usefulness of MLPA in a quantitative description of microbial shifts. © 2008 Published by Elsevier B.V. 1. Introduction The human oral cavity harbors a multifarious group of microorgan- isms that forms a complex community and occupies diverse specific niches and microenvironments. The resident oral microflora is sustained in an apparent state of balance or microbial homeostasis, once microorganisms are established in the mouth (Marsh, 2003a). Environ- mental perturbations change the dynamic ecology of the resident, evoking microbial shifts in terms of concentrations and activities, which at times may result in the development of cariogenic and periodontal diseases (Marsh, 2000); 1994). Dental plaque, a surface bound community which is regarded as a biofilm is of particular importance in the ecology of the oral microbiota. The formation of plaque or growth of biofilm on dental surfaces by rapid colonization of bacteria follows a particular microbial succession, which is largely dependent on the hosts genotype, quality of the immune system, diet, general hygiene and health conditions (Marsh, 2003a; Palmer et al., 2007). Growing in a biofilm gives certain advantages to the resident microflora (Scheie and Petersen, 2004). The biofilm life style enables bacteria to develop mechanisms that minimize the effect of antimicrobials and the human immune defense system (Gilbert et al., 1997; Mah and O'Toole, 2001). About half of the more than 700 bacteria species inhabiting the oral cavity are culturable, though this assumption is made by studying mainly sub-gingival samples (Aas et al., 2005; Pratten et al., 2003). Traditional culturing methods and biochemical assays thus do not allow to fully characterize the inhabitants of the oral cavity. Instead, molecular techniques provide the ideal tools for identification and characterization of bacteria that are hitherto undiscovered and most of the traditionally uncultivable bacteria. Current determination of the microbial etiology of dental diseases which utilizes both the culturing and molecular methods is far from being adequate (Marsh, 2003b). Similarly, quantification of the bacteria in biofilms is a daunting task. The change in the dynamics of the resident flora due to changes in the environment poses a challenge for accurate determination of the spatial and temporal abundance of the species. In-vitro models show ways of evaluating the shift in populations associated with the onset of common oral diseases (Dalwai et al., 2006). Real-time PCR based methods such as the Taq-man system and qPCR and checkerboard DNA–DNA hybridization are shown to quantify oral Journal of Microbiological Methods 75 (2008) 558–565 ⁎ Corresponding author. Academic Centre for Dentistry Amsterdam, Louwesweg 1, 1066EA Amsterdam, The Netherlands. Tel.: +31 205188596; fax: +31 206692881. E-mail address: z.terefework@acta.nl (Z. Terefework). 0167-7012/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.mimet.2008.08.012 Contents lists available at ScienceDirect Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth