Short Communication Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food M. Guarddon a , J.M. Miranda a , J.A. Rodríguez b , B.I. Vázquez a , A. Cepeda a , C.M. Franco a, ⁎ a Laboratorio de Higiene, Inspección y Control de Alimentos, Departamento de Química Analítica, Nutrición y Bromatología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002-Lugo, Spain b Area Académica de Química, Universidad Autónoma del Estado de Hidalgo, Carretera Pachuca-Tulancingo, Kilómetro 4.5, Ciudad Universitaria, 42074, Pachuca de Soto, Hidalgo, Mexico abstract article info Article history: Received 8 November 2010 Received in revised form 21 January 2011 Accepted 21 February 2011 Keywords: Tetracycline resistance Food PCR tetA tetB A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in Gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7 log cfu/mL) for tetB and 5 × 10 2 cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline- resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r = 0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r = 0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples. © 2011 Elsevier B.V. All rights reserved. 1. Introduction The emergence of antimicrobial-resistant bacterial pathogens after several decades of antimicrobial use is becoming a major concern (Martinez et al., 2009). During animal production, large amounts of antimicrobials are used for disease control. As a consequence, favorable conditions have been created for the emergence and dissemination of antimicrobial-resistant bacteria (Aarestrup, 2005; Miranda et al., 2007, 2008). Accordingly, many studies have reported on the risks posed by antibiotic-resistant bacteria on human health, which is a result of the untreatable diseases caused by some bacterial pathogens (Levy, 1998; Levy and Marshall, 2004; White, 2006). The use of antimicrobials as growth promoters has been prohibited in the European Union (EU) (Regulation (EC) 1831/2003). However, in many countries, their use is still allowed. Moreover, antimicrobials are also employed for animal therapy or prophylaxis (Aarestrup, 2005; Guardabassi and Courvalin, 2006). Tetracyclines are broad-spectrum agents that show activity against a wide range of Gram-positive and Gram-negative bacteria, and their favorable antimicrobial properties have extended their use in the therapy of animal infections. In fact, the widespread use of tetracyclines in clinical practice has been responsible for the selection of resistant organisms to this antimicrobial family (Chopra and Roberts, 2001). Antimicrobial-resistance genes are transmissible between different bacteria and from bacteria carried by food-producing animals to human bacteria (Aarestrup, 2005). This ability depends on the presence and transfer of resistance genes among bacteria, mutations and selection pressure to keep antimicrobial-resistance genes in a bacterial popula- tion (Bryan et al., 2004). Thus, tetracycline resistance (tet) genes, which are often associated with mobile elements, have been spread among bacterial species. Currently, over 40 tet genes have been identified (Roberts, 2005). These antibiotic resistance genes can be studied by real- time polymerase chain reaction (qPCR), which is a rapid, effective and handy tool, compared to conventional methods that are used in microbiological research (Martinez et al., 2007). Quantitative PCR represents an innovative and specific technique (Klein, 2002; Rodriguez-Lazaro et al., 2004) that is widely used in food microbiology (Pennacchia et al., 2009). However, most of the studies regarding tetracycline resistance in microorganisms have employed clinical samples or bacteria isolated from different matrices that can International Journal of Food Microbiology 146 (2011) 284–289 ⁎ Corresponding author at: Laboratorio de Higiene, Inspección y Control de Alimentos, Facultad de Veterinaria pabellón 4 p.b., Campus Universitario 27002-Lugo, Spain. Tel.: +34 982285900x22454; fax: +34 982254592. E-mail address: carlos.franco@usc.es (C.M. Franco). 0168-1605/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2011.02.026 Contents lists available at ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro