Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products
Introduction
Lactococcus lactis is one of the best known and characterized species of Lactic Acid Bacteria (LAB). These bacteria are present in the natural environment, including products of spontaneous milk or plant fermentation. They play an important role in the production of various dairy products but are also used as food additives and animal feed. In addition, lactococci are also responsible for flavor formation through their proteolytic and amino acid conversion pathways (Rattanachaikunsopon and Phumkhachorn, 2010).
The common use of antibiotics in medicine and as “growth promoters” in animal breeding has caused a significant increase in the number of strains resistant to antibiotics, including among LAB. Tetracyclines are one example of such extensively used antibiotics. They are broad-spectrum antibiotics active against both Gram-positive and Gram-negative bacteria that act at the ribosomal level to interfere with bacterial protein synthesis. Tetracyclines have been widely used in livestock farming as well as in the prophylactic and therapeutic treatment of human and animal infections. Unfortunately, this prevalent use of tetracyclines has led to an increase in antibiotic resistance. The major mechanisms of tetracycline resistance involve efflux pumps, ribosomal protection proteins, and direct enzymatic drug inactivation. To date, more than 40 tetracycline-resistance genes have been identified and characterized, and the best known are tet(M), tet(S) and tet(O) (van Hoek et al., 2011). These genes are often located on mobile elements, such as plasmids and transposons, and may therefore be easily transferred between bacteria (Clewell et al., 1995, Rice, 1998).
The concomitant presence of antibiotics and bacteria growing at high densities enables the spread of antibiotic resistance among microorganisms. LAB, which are an element of the gastrointestinal microbiota, are potentially vulnerable to acquired antibiotic resistance. Furthermore, genes conferring antibiotic resistance can be easily transferred between pathogenic, potentially pathogenic and commensal bacteria (Delgado et al., 2005, Devirgiliis et al., 2011, Mathur and Singh, 2005). Based on the above-described observations, it has been hypothesized that the gastrointestinal bacteria, including commensals, may be reservoirs of antibiotic-resistance genes (Salyers et al., 2004).
The increase in antibiotic resistance among microorganisms has been recognized as one of the most serious public health problems in the European Union (ECDC/EMEA, 2009). Therefore, it is crucial to estimate the level of antibiotic resistance of lactic acid bacteria and assess the role of LAB as a source of antibiotic-resistance genes. Thus, the aim of this study was to evaluate the presence of LAB strains resistant to antibiotics in samples isolated from Polish raw milk and fermented artisanal products as well as to molecularly characterize the L. lactis strains resistant to tetracycline.
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Bacterial strains and growth conditions
Five-hundred LAB isolates were recovered at the turn of the century from samples of Polish artisanal dairy products and raw milk from cows, sheep and goats collected from individual farms and local food markets were used in this study. The isolates were grown in M17 broth (Difco, Detroit, MI, USA) supplemented with 0.5% (wt/vol) glucose (POCH, Gliwice, Poland) (GM17) at 30 °C for 24 to 48 h. Agar plates were prepared by adding 1.5% (wt/vol) agar (Merck, Darmstadt, Germany) to the liquid medium.
Isolation and taxonomic identification of antibiotic-resistant strains
Tetracycline-resistant (TetR) and vancomycin-resistant (VanR) isolates were recovered from various regions of Poland (data not shown). Of the 500 LAB isolates that were tested in the study, only seven (IBB11, IBB28, IBB160, IBB161, IBB224, IBB477, and IBB487) were able to grow on plates with tetracycline, whereas two isolates (IBB62 and IBB64) grew on plates with vancomycin. None of the isolates grew on media with the other tested antibiotics.
Six TetR isolates namely IBB28, IBB160, IBB161,
Discussion
Antibiotics play an important role in decreasing morbidity and mortality associated with bacterial infections and have a significant impact on the success of medicine. Additionally, they are also used as therapeutic agents and animal growth promoters and in agriculture for the control of plant diseases (Ammor et al., 2007, Wegener, 2003). This huge amount of used antibiotics has significantly affected the bacterial environment and has led to the selection of new antibiotic-resistant strains (
Acknowledgments
This work was performed in the frame of the EU-founded project “Assessment and Critical Evaluation of Antibiotic Resistance Transferability in Food Chain” (ACE-ART; CT-2003-506214) of the 6th Framework Program and was also supported by own statutory funds of IBB PAS that stands for our Institute -Institute of Biochemistry and Biophysics of Polish Academy of Sciences. This work was also partly financed by European Funds Portal Innovative Economy "Centre of medicinal product biotechnology.
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- 1
Both first authors equally contributed to this paper.
- 2
Present address: Department of Bacterial Genetics, Institute of Microbiology, Warsaw University, Miecznikowa 1, 02–096 Warsaw, Poland.