Research Article Molecular Identification and Quantification of Tetracycline and Erythromycin Resistance Genes in Spanish and Italian Retail Cheeses Ana Belén Flórez, 1 Ángel Alegría, 1,2 Franca Rossi, 2 Susana Delgado, 1 Giovanna E. Felis, 2 Sandra Torriani, 2 and Baltasar Mayo 1 1 Departamento de Microbiolog´ ıa y Bioqu´ ımica, Instituto de Productos L´ acteos de Asturias (IPLA-CSIC), Paseo R´ ıo Linares s/n, Villaviciosa, 33300 Asturias, Spain 2 Dipartimento di Biotecnologie, Universit` a Degli Studi di Verona, Strada Le Grazie, 15, 37134 Verona, Italy Correspondence should be addressed to Baltasar Mayo; baltasar.mayo@ipla.csic.es Received 4 July 2014; Revised 21 August 2014; Accepted 27 August 2014; Published 11 September 2014 Academic Editor: Gundlapally S. Reddy Copyright © 2014 Ana Bel´ en Fl´ orez et al. his is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Large antibiotic resistance gene pools in the microbiota of foods may ultimately pose a risk for human health. his study reports the identiication and quantiication of tetracycline- and erythromycin-resistant populations, resistance genes, and gene diversity in traditional Spanish and Italian cheeses, via culturing, conventional PCR, real-time quantitative PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). he numbers of resistant bacteria varied widely among the antibiotics and the diferent cheese varieties; in some cheeses, all the bacterial populations seemed to be resistant. Up to eight antibiotic resistance genes were sought by gene-speciic PCR, six with respect to tetracycline, that is, tet(K), tet(L), tet(M), tet(O), tet(S), and tet(W), and two with respect to erythromycin, that is, erm(B) and erm(F). he most common resistance genes in the analysed cheeses were tet(S), tet(W), tet(M), and erm(B). he copy numbers of these genes, as quantiied by qPCR, ranged widely between cheeses (from 4.94 to 10.18log 10 /g). DGGE analysis revealed distinct banding proiles and two polymorphic nucleotide positions for tet(W)-carrying cheeses, though the similarity of the sequences suggests this tet(W) to have a monophyletic origin. Traditional cheeses would therefore appear to act as reservoirs for large numbers of many types of antibiotic resistance determinants. 1. Introduction Antibiotic resistance (AR) is a natural phenomenon, the appearance of which predates the clinical use of antibiotics [1, 2]. Unfortunately, the widespread use and misuse of antibiotics in clinical and nonclinical environments for more than seven decades have provided optimal conditions for the appearance, mobilization, and concentration of highly eicient resistance systems in bacteria [3]. he transfer of AR genes into human and animal pathogens could ultimately lead to a failure of antibiotic therapy [4]. Mobilization among bacterial species is facilitated by AR genes being commonly located on mobile genetic elements such as transposons and plasmids, which have high horizontal transfer capacity [5]. he presence of antibiotics in the environment not only provides a positive selection for resistant pathogens, but also exerts an evolutive pressure on components of the commensal microbiota [6]. Under these conditions, the com- mensal bacteria in food could become a reservoir for AR determinants that could then further be disseminated via the food chain [7–9]. Determining the prevalence of AR genes in a given envi- ronment, and their characterization, requires the isolation and identiication of the resistant bacteria, followed by a molecular analysis of their AR determinants [10–12]. he identiication and quantiication of AR genes directly in envi- ronmental samples, that is, without culturing biases, would be useful [13, 14]. Culture-independent analysis is also faster and more accurate than culture-based methods. Indeed, several AR identiication and AR gene quantiication techniques Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 746859, 10 pages http://dx.doi.org/10.1155/2014/746859