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Projects / Programmes source: ARRS

Resistomes of probiotic and starter cultures as potential risk factors for the spread of antibiotic resistance

Research activity

Code Science Field Subfield
4.02.04  Biotechnical sciences  Animal production  Processing of animal raw materials 

Code Science Field
B230  Biomedical sciences  Microbiology, bacteriology, virology, mycology 
Keywords
starter cultures, probiotics, food chain, antibiotic resistance, resistome, dairy products, lactic acid bacteria
Evaluation (rules)
source: COBISS
Researchers (15)
no. Code Name and surname Research area Role Period No. of publications
1.  11150  PhD Bojana Bogovič Matijašić  Animal production  Principal Researcher  2019 - 2022  362 
2.  15659  PhD Andreja Čanžek Majhenič  Animal production  Researcher  2019 - 2022  227 
3.  30378  PhD Majda Golob  Veterinarian medicine  Researcher  2019 - 2022  167 
4.  30755  PhD Sandra Janežič  Microbiology and immunology  Researcher  2019 - 2022  131 
5.  37544  PhD Aleksander Mahnič  Microbiology and immunology  Researcher  2019 - 2022  51 
6.  25516  PhD Petra Mohar Lorbeg  Animal production  Researcher  2019 - 2022  98 
7.  28205  Tanja Obermajer    Technician  2019 - 2022  48 
8.  11133  PhD Matjaž Ocepek  Veterinarian medicine  Researcher  2019 - 2022  451 
9.  36376  PhD Diana Paveljšek  Animal production  Researcher  2019 - 2022  28 
10.  08857  PhD Irena Rogelj  Animal production  Researcher  2019 - 2022  703 
11.  50598  PhD Vita Rozman  Animal production  Junior researcher  2019 - 2022  30 
12.  12278  PhD Maja Rupnik  Microbiology and immunology  Researcher  2019 - 2022  627 
13.  33511  PhD Valerija Tkalec  Microbiology and immunology  Researcher  2019 - 2022  49 
14.  31910  PhD Primož Treven  Animal production  Researcher  2019 - 2022  62 
15.  12682  PhD Irena Zdovc  Veterinarian medicine  Researcher  2019 - 2022  443 
Organisations (3)
no. Code Research organisation City Registration number No. of publications
1.  0406  University of Ljubljana, Veterinary Faculty  Ljubljana  1627139  9,592 
2.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  63,013 
3.  3334  National Laboratory of Health, Environment and Foodstaffs  Maribor  6489087  3,933 
Abstract
The intensive use of antibiotics in human and veterinary medicine in the past 50 years has brought the problem of resistance to antibiotics (AR) in pathogenic microorganisms and reduced the effectiveness of infections’ treatment which the World Health Organisation declared to be one of the greatest threats to health. It is well known that the antibiotic resistance genes (ARGs) can be horizontally transferred among bacteria of different species, and pathogenic bacteria with AR occur along the entire agro-food chain. The fact that the commensal bacteria in food can present a reservoir of ARG have begun to draw attention of scientists years ago, but until recently there were not so advanced and powerful molecular methods and technologies available that enable the disclosure of whole bacterial genomes and microbiomes of complex samples, including the "resistome" - collection of all AR genes present in a given environment.The proposed study will be focused on the bacterial representatives of starter cultures and probiotics, which are intentionally added to the food chain, as well as on their whole genomic sequences (WGS) - either publicly available or those that will be obtained in this study. Using next generation sequencing (Illumina technology), we will acquire the WGSs of commercial or own bacterial isolates, representatives of starter and probiotic cultures, and thus enrich the public databases. In the known or newly acquired WGS, we will in silico identify the ARGs, mutations and mobile elements, by comparative genomic analysis, and determine the matching of the in silico AR predictions with the results of phenotypic detection of sensitivity to selected antibiotics. The latter is important from the standpoint of assessing the nature (intrinsic or acquired) of the observed phenotypic resistance and possible improvement of criteria (MIC cut-off) for AR in lactic acid bacteria and bifidobacteria. We will set up and validate the protocols based on polymerase chain reaction (PCR, qPCR) for the targeted screening of the resistomes of probiotic products and fermented foods, produced with commercial starter cultures or without them. Furthermore, selected food samples will be examined by metagenomic sequencing in order to identify more ARGs and determine the relative abundances of these ARGs, and to asses the contribution of added starter and probiotic cultures to the resistomes of investigated food samples. The approach presented in the submitted project, which includes in silico examination of whole bacterial genomes, metagenomic sequencing of complex food samples, and quantitative support by real-time PCR (qPCR) will enable efficient detection of these features in different resistomes associated with foods, and better assessment of the risk of transmission of ARGs along the food chain. Thus we expect to cover the gap between previous studies, which were based on phenotypic testing and detection of a limited number of well-known targets, and among those in the coming years, when the use of the most powerful tools for metagenomic analyses is expected to spread well into the food safety area.
Significance for science
New approaches to assessment of antibiotic resistance (AR) (comparative genomics, resistome approach), will be implemented in the frame of this project, which is important from the standpoint of research excellence of participating institutions - the University of Ljubljana and NLZOH. Despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. More advanced approaches have recently been introduced also in the field of AR in medically important bacteria, and more slowly for commensal bacteria. Some previous EU projects, such as ACE-ART (6FP) - Assessment and critical evaluation of antibiotic resistance transferability in food chain (2004-2007) and ARBFOOD - Antibiotic resistance genes in food: Molecular identification and transfer between micro-organisms (2001-2004) drew attention to the problem of commensal bacteria in food chain as reservoirs for AR, but at that time, the high-throughput molecular methods that allow disclosure of whole bacterial genomes and microbiomes of complex samples including food have not yet been available. In this project proposal we focused on the representatives of bacterial starter cultures and probiotics, which are intentionally added to the food chain, and their whole genomic sequences (WGS) - either publicly available or those that will be obtained in this study. More comprehensive approach towards detection of AR in the representatives of starter cultures and probiotics, which will be based on in silico analysis of WGS and the identification of genetic determinants associated with AR, will enable efficient detection of these features in different resistomes associated with the food chain, and better assessment of the risk of transmission of ARGs along the food chain. Methods and tools which will be appropriately validated also on the samples from the food chain (starter and probiotic cultures, food, probiotic products ...) will also be useful for other researchers. In addition, we will contribute to the public databases new genomic sequences of selected representatives of lactic acid bacteria and bifidobacteria, which can be used in research by other researchers. Approach with the most advanced tools - the so called full metagenomic "shotgun" sequencing of entire microbiomes or targeted metagenomic sequencing of the entire resistomes using last generation capture platforms specifically developed to analyze resistomes – have also been included in the proposed program. Considering the financial frame of this call, we will not be able to include a large number of samples, but we expect to cover the gap between previous studies, which were based on phenotypic testing and detection of a limited number of well-known targets, and among those in the coming years, when the use of the most powerful tools for metagenomic analyses is expected to spread well into the food safety area, as it will become more accessible.
Significance for Slovenia
New approaches to assessment of antibiotic resistance (AR) (comparative genomics, resistome approach), will be implemented in the frame of this project, which is important from the standpoint of research excellence of participating institutions - the University of Ljubljana and NLZOH. Despite the claim that the food chain plays a key role in the spread of antibiotic resistance, metagenomic analysis has scarcely been used to investigate food systems. More advanced approaches have recently been introduced also in the field of AR in medically important bacteria, and more slowly for commensal bacteria. Some previous EU projects, such as ACE-ART (6FP) - Assessment and critical evaluation of antibiotic resistance transferability in food chain (2004-2007) and ARBFOOD - Antibiotic resistance genes in food: Molecular identification and transfer between micro-organisms (2001-2004) drew attention to the problem of commensal bacteria in food chain as reservoirs for AR, but at that time, the high-throughput molecular methods that allow disclosure of whole bacterial genomes and microbiomes of complex samples including food have not yet been available. In this project proposal we focused on the representatives of bacterial starter cultures and probiotics, which are intentionally added to the food chain, and their whole genomic sequences (WGS) - either publicly available or those that will be obtained in this study. More comprehensive approach towards detection of AR in the representatives of starter cultures and probiotics, which will be based on in silico analysis of WGS and the identification of genetic determinants associated with AR, will enable efficient detection of these features in different resistomes associated with the food chain, and better assessment of the risk of transmission of ARGs along the food chain. Methods and tools which will be appropriately validated also on the samples from the food chain (starter and probiotic cultures, food, probiotic products ...) will also be useful for other researchers. In addition, we will contribute to the public databases new genomic sequences of selected representatives of lactic acid bacteria and bifidobacteria, which can be used in research by other researchers. Approach with the most advanced tools - the so called full metagenomic "shotgun" sequencing of entire microbiomes or targeted metagenomic sequencing of the entire resistomes using last generation capture platforms specifically developed to analyze resistomes – have also been included in the proposed program. Considering the financial frame of this call, we will not be able to include a large number of samples, but we expect to cover the gap between previous studies, which were based on phenotypic testing and detection of a limited number of well-known targets, and among those in the coming years, when the use of the most powerful tools for metagenomic analyses is expected to spread well into the food safety area, as it will become more accessible.
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