Identification and characterization of tetracycline resistance in Lactococcus lactis isolated from Polish raw milk and fermented artisanal products

https://doi.org/10.1016/j.ijfoodmicro.2015.07.009Get rights and content

Abstract

To assess the occurrence of antibiotic-resistant Lactic Acid Bacteria (LAB) in Polish raw milk and fermented artisanal products, a collection comprising 500 isolates from these products was screened. Among these isolates, six strains (IBB28, IBB160, IBB161, IBB224, IBB477 and IBB487) resistant to tetracycline were identified. The strains showing atypical tetracycline resistance were classified as Lactococcus lactis: three of them were identified as L. lactis subsp. cremoris (IBB224, IBB477 and IBB487) and the other three (IBB28, IBB160, IBB161) were identified as L. lactis subsp. lactis. The mechanism involving Ribosomal Protection Proteins (RPP) was identified as responsible for tetracycline resistance. Three of the tested strains (IBB28, IBB160 and IBB224) had genes encoding the TetS protein, whereas the remaining three (IBB161, IBB477 and IBB487) expressed TetM. The results also demonstrated that the genes encoding these proteins were located on genetic mobile elements. The tet(S) gene was found to be located on plasmids, whereas tet(M) was found within the Tn916 transposon.

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.

Section snippets

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.

References (56)

  • A.P. Roberts et al.

    A modular master on the move: the tn916 family of mobile genetic elements

    Trends Microbiol.

    (2009)
  • A.A. Salyers et al.

    Human intestinal bacteria as reservoirs for antibiotic resistance genes

    Trends Microbiol.

    (2004)
  • H.C. Wegener

    Antibiotics in animal feed and their role in resistance development

    Curr. Opin. Microbiol.

    (2003)
  • M.S. Ammor et al.

    Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

    J. Mol. Microbiol. Biotechnol.

    (2008)
  • R.I. Aminov et al.

    Molecular ecology of tetracycline resistance: development and validation of primers for detection of tetracycline resistance genes encoding ribosomal protection proteins

    Appl. Environ. Microbiol.

    (2001)
  • D.G. Anderson et al.

    Simple and rapid method for isolating large plasmid DNA from lactic streptococci

    Appl. Environ. Microbiol.

    (1983)
  • R. Barrangou et al.

    Identification and characterization of Leuconostoc Fallax strains isolated from an industrial sauerkraut fermentation

    Appl. Environ. Microbiol.

    (2002)
  • H.C. Birnboim et al.

    A rapid alkaline extraction procedure for screening recombinant plasmid DNA

    Nucleic Acids Res.

    (1979)
  • J. Boguslawska et al.

    Intra- and interspecies conjugal transfer of Tn916 like elements from Lactococcus lactis in vitro and in vivo

    Appl. Environ. Microbiol.

    (2009)
  • S.L. Bronzwaer et al.

    A European study on the relationship between antimicrobial use and antimicrobial resistance

    Emerg. Infect. Dis.

    (2002)
  • I. Chopra et al.

    Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance

    Microbiol. Mol. Biol. Rev.

    (2001)
  • S. Delgado et al.

    Antibiotic susceptibility of Lactobacillus and Bifidobacterium species from the human gastrointestinal tract

    Curr. Microbiol.

    (2005)
  • C. Devirgiliis et al.

    Identification of tetracycline- and erythromycin-resistant Gram-positive cocci within the fermenting microflora of an Italian dairy food product

    J. Appl. Microbiol.

    (2010)
  • C. Devirgiliis et al.

    Antibiotic resistance determinants in the interplay between food and gut microbiota

    Gene Nutr.

    (2011)
  • ECDC/EMEA

    The Bacterial Challenge: Time to React

    ECDC/EMEA Joint Technical Report

    (2009)
  • EFSA

    Opinion of the scientific panel on additives and products or substances used in animal feed on the updating of the criteria used in assessment of bacteria for resistance to antibiotics of human or veterinary importance

    EFSA J.

    (2005)
  • EFSA

    Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance

    EFSA J.

    (2012)
  • EFSA

    Scientific opinion on the maintenance of the list of QPS biological agents intentionally added to food and feed (2013 update)

    EFSA J.

    (2013)
  • Cited by (23)

    • Antibiotic resistance in wild and commercial non-enterococcal Lactic Acid Bacteria and Bifidobacteria strains of dairy origin: An update

      2022, Food Microbiology
      Citation Excerpt :

      Several resistance determinants have been found in Lc. lactis (Table 1), indeed both L. lactis IPLA 31008 and IPLA 31009 were positive for a plasmid associated with a tetM gene (Flórez et al., 2008), which was identical to the tetM encoded by the conjugative transposon Tn916, i.e. a conjugative transposon that has showed the possibility of being transferred between Gram-positive and Gram-negative organisms (Bertram et al., 1991); also Zycka-Krzesinska et al. (2015) found tetM gene within the Tn916 transposon, whereas tetS gene was found to be located on plasmids in Lc. lactis (Table 4).

    • Microorganisms from starter and protective cultures - Occurrence of antibiotic resistance and conjugal transfer of tet genes in vitro and during food fermentation

      2022, LWT
      Citation Excerpt :

      On the other hand, study by Dušková et al. (2020) showed that among the Lactobacillus strains from starter cultures, the highest MIC values were for aminoglycosides. However, the results of phenotypic antibiotic resistance patterns of other genera are not in agreement with other studies, which showed the highest percentage of strains with high MIC for erythromycin, while few strains showed high MIC for tetracycline (Federici et al., 2014; Zycka-Krzesinska, Boguslawska, Aleksandrzak-Piekarczyk, Jopek, & Bardowski, 2015). This may be due to differences between individual strains of the same genus or species, or to fluctuations in the expression of the genes responsible for revealing the phenotypic trait.

    • Nafcillin degradation by heterogeneous electro-Fenton process using Fe, Cu and Fe/Cu nanoparticles

      2020, Chemosphere
      Citation Excerpt :

      Its introduction in these systems could be caused by the poor care of the population by eliminating expired medication; since these are thrown directly into the sewerage network and the wastewater produced by hospitals, pharmaceuticals and livestock industries (Sanganyado and Gwenzi, 2019). Although concentrations have been detected in the order of micro or nano grams, this small amount is sufficient to contribute to the multi-resistance of microorganisms (Zycka-Krzesinska et al., 2015). For example, an average concentration of 28.3 ng/L β-lactams was detected in a river in China (Lei et al., 2019).

    • Detection in raw cow's milk of coliform bacteria - reservoir of antibiotic resistance

      2018, LWT
      Citation Excerpt :

      Nowadays, a significant increase in the number of strains resistant to antibiotics is being widely observed, what results from the common use of antibiotics both in medicine and as “growth promoters”(Zycka-Krzesinska, Boguslawska, Aleksandrzak-Piekarczyk, Jopek, & Bardowski, 2015).

    View all citing articles on Scopus
    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.

    View full text