1. Is Your Food Still Safe to Eat?

2. 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

3. Genesis of the project- Antibiotic Resistance

4. Genesis of the project- Why Lactobacillus sp.?

5. 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

6. Genesis of the project- Why Lactobacillus sp.?

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

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

9. 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.

10. 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.

11. 7 strains 7 strains

12. Results
Occurrence of AR genes
13 erm(B) genes

13. Results
Occurrence of AR genes
10 cat- TC genes

14. Results
Occurrence of AR genes
0 tet(W) genes

15. 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

16. 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

18. 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.

19. 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