Is Your Food Still Safe to Eat?

Is Your Food Still Safe to Eat? Occurrence of antibiotic resistance genes within Lactobacillus sp. isolated from food products Karolina Kacynel University of Warmia and Mazury in Olsztyn, Poland

Genesis of the project- Antibiotic Resistance

Genesis of the project- Why Lactobacillus sp.? http://i0.kym-cdn.com/photos/images/original/000/533/539/5b7.jpg

Genesis of the project- Why Lactobacillus sp.? 152 species 17 subspecies No 1 LAB Fig. Long bacillli of L. bulgaricus Fig. Short bacilli of L. fermentum Fig. Coccus forms of L. lactis Fig. . V-shaped L. curvatus

Genesis of the project- Why Lactobacillus sp.?

Lactobacillus sp. – safe bacteria? Broad knowledge of physiology, metabolism and genetics

Lactobacillus sp. – safe bacteria? reservoirs of AR genes Overuse antibiotics Food chain HTG

Aim of the study (+ M&M) 1 2 3 Assessment of the prevalence of AR genes Collection of Lactobacillus sp. isolated from: fermented dairy, meat and vegetable products produced by traditional and industrial methods Systematic identification of Lactobacillus sp.

Systematic identification of Lactobacillus sp. Results Systematic identification of Lactobacillus sp. 1. Preliminary identification of isolated bacterial strains Macroscopic methods growth in liquid + solid medium Microscopic methods cell morphology (Gram staining) 2. PCR identification 21 strains of G(+) bacilli 18 strains of Lactobacillus sp.

7 strains 7 strains

Results Occurrence of AR genes 13 erm(B) genes

Results Occurrence of AR genes 10 cat- TC genes

Results Occurrence of AR genes 0 tet(W) genes

Discussion 15 out of 18 Lactobacillus sp. strains had at least 1 AR gene. In 8 strains (44% of cases)  multi-drug resistance L. plantarum  57% of strains were multiresistant L. brevis  only 28%. A relationship between the frequencies of the genes and the origin of the strains (the sort of product and the production method) was not observed. Origin of the strains Genes

Conclusions The received data from this research: can lead to better view of breakpoints in current microbiological categorisation of genetically safe bacteria strains. can help to assess the danger caused by the AR occurrence among food-associated bacteria. danger  human and animal health & life reveal the seriousness of the AR problem Karolina Kacynel University of Warmia and Mazury in Olsztyn, Poland Department of Biology and Biotechnology kkacynel@interia.eu

PCR reaction mixture composition The thermal profile of PCR Material & Methods  PCR reaction mixture composition dNTP 200 µM each Primers 1 & 2 0,1 µM each Polimerase Taq 1,25 U 1x Taq bufore + KCl i + MgCl2 2 mM DNA 20‑40 ng/µl The thermal profile of PCR Step Genus/ species affiliation AR genes identification Initial denaturation  94°C /5 min.  95°C /5 min. Denaturation 35 cycles 95°C/30 s. 40 cycles Annealing 2 min., temp. depending on the identification 30 s., temp. depending on the identification Extension 60°C/2 min. 72°C/45 s. Final extension 60°C /5 min. 72°C /7 min.

Material & Methods  Tab. Genus and species specific primers used in the study and the annealing temperature in PCR Type of identification Primers DNA sequence Annealing tempperature [°C] Product size [pz] Lactobacillus LbLMA1-rev R16-1 5’- CTCAAAACTAAACAAAGTTTC-3’ 5’-CTTGTACACACCGCCCGTCA-3’ 55 250 L. plantarum Lpla-3 Lpla-2 5’-ATTCATAGTCTAGTTGGAGGT-3’ 5’-CCTGAACTGAGAGAATTTGA-3’ 60 248 L. brevis Bre-16S.L Bre-ITS.R 5’-GTGAGATAACCTTCGGGAGT-3’ 5’-GGTCACTTCGTGATCGTCAA-3’ 62 316 Tab. AR gene primers used in PCR Antibiotic (AR gene) Primers DNA sequence Annealing tempperature [°C] Product size [pz] Erytromycyna (erm(B)) ermBf ermBr 5’-CATTTAACGACGAAACTGGC-3’ 5’-GGAACATCTGTGGTATGGCG-3’ 55 425 Chloramfenikol (cat-TC) cat-TCf cat-TCr 5’-CAATAGCGACGGAGAGTTAGG-3’ 5’-AATCCTGCATGATAACCATCAC-3’ 384 Tetracyklina (tet(W)) tetWf tetWr 5’-GAGAGCCTGCTATATGCCAGC-3’ 5’-GGGCGTATCCACAATGTTAAC-3’ 64 168