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

The intensive use of antimicrobials in human and veterinary medicine in the past 50 years has brought the problem of resistance 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. 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. By comparative genomic analysis, we will their whole genome sequences (WGSs),  we will in silico identify the ARGs, mutations and mobile elements, and determine the matching of the in silico AR predictions with the results of phenotypic detection. 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 this study, will enable efficient detection of ARGs in different resistomes associated with foods, and better assessment of the risk of transmission of ARGs along the food chain.


Project identifier:                        J4-1769
Type of the project:                    Basic research
Duration of the project:             1.7.2019-30.6.2022   
Title:                                              Resistomes of probiotic and starter cultures as potential risk factors for the spread of antibiotic resistance
Financed by:                                Slovenican Research Agency (ARRS)
Participating organisations:    University of Ljubljana, Biotechnical Faculty, Institute of dairy science and probiotics
                                                       National laboratory of health, environment and food
                                                       University of Ljubljana, Veterinary Faculty, Institute of Microbiology and Parasitology
Project leader:                             Sen. Res. Fellow Bojana Bogovič Matijašić, PhD, B.Sc. Food Technol.
University of Ljubljana, Biotechnical Faculty, Institute of dairy science and probiotics
Sen. Res. Fellow Bojana Bogovič Matijašić, PhD, B.Sc. Food Technol.– project leader;  Prof. Irena Rogelj, PhD, B.Sc. Food Technol.; Assoc. Prof. Andreja Čanžek Majhenič, PhD, B.Sc. Food Technol.; Assist. Petra Mohar Lorbeg, PhD, B.Sc. Food Technol.; Assist. Primož Treven, PhD, B.Sc. Biochem; Diana Paveljšek, PhD, univ. dipl. inž. živil. tehnol.; Tanja Obermajer, B.Sc. Biol.; Vita Rozman, M.Sc. Biotechnol. – Early Stage Researcher

National laboratory of health, environment and food

prof. Maja Rupnik, PhD, B.Sc. Biol.; Assist. Prof. Sandra Janežič, PhD, B.Sc. Microbiol.; Assist. Valerija Tkalec, PhD, B.Sc. Microbiol.; Aleksander Mahnič, PhD, B.Sc. Microbiol.

University of Ljubljana, Veterinary Faculty, Institute of Microbiology and Parasitology
Assist. Prof. Irena Zdovc, PhD, DVM; Matjaž Ocepek, PhD, DVM; Majda Golob, DVM


The intensive use of antibiotics in human and veterinary medicine in the past 50 years has brought the problem of resistance to antibiotics in pathogenic microorganisms and reduced the effectiveness of infections’ treatment. Considering the rapid emergence of antibiotic-resistant bacterial pathogens coupled with a lack of new antibiotics in the development pipeline, resistance and its consequences have become a major global health concern. 25,000 deaths in the European Union (EU) every year are estimated to be caused due to infections by multidrug-resistant bacteria. Furthermore, the failure of antimicrobial therapies also strongly affect healthcare systems and society, as healthcare expenditures and productivity losses are estimated to reach €1.5 billion/year in the EU1. It is well known that the pathogenic bacteria with antibiotic resistance (AR) occur along the entire food chain, from livestock, food industry, food, humans, to the waste associated with animal husbandry and food industry2,3. One of the main sources of this problem is the intensive rearing of animals intended for consumption, mainly due to the use of antibiotics in sub-therapeutic dosages, which promote the development of antibiotic-resistant bacteria. In order to reduce the spread of AR, the use of antibiotics in animal feed, for prevention and growth promotion, was banned in the EU already in 2006. In addition, intensive activities aimed at reducing the use of antibiotics in human4,5 and in veterinary medicine6 are taking place in the EU.  

It is well known that under certain environmental conditions (selection pressure of antibiotics, dense colonisation of environment with microorganisms, etc.) genes for antibiotic resistance (ARG) can be horizontally transferred among bacteria of different species or genera. The mechanisms of horizontal transfer are quite well investigated for particular antibiotics, especially those associated with pathogenic bacteria which are often involved in nosocomial infections. Migration of pathogenic bacteria that have developed AR to several antibiotics and pose a threat to health throughout the food chain is a problem, which has long been recognized and studied7. The fact that the commensal bacteria in the food chain can present a reservoir of ARG for dissemination, however, have begun to draw attention of scientists only in recent time8,9.

Lactic acid bacteria (LAB) and bifidobacteria are intentionally introduced into the food chain through starter cultures for food, probiotics for humans or feed additives. The concept of Qualified presumption of Safety (European Food Safety Authority-EFSA), which established criteria for the safety of bacteria used in the EU in the food chain (for food or feed) contains a requirement for the absence of acquired ARG to clinically important antibiotics10,11. Already since 2001, when the first guidelines for determining the nature of the resistance to clinically important antibiotics in micro-organisms, which are used as feed additives or as production organisms, were set up (SCAN - Scientific Committee on Animal Nutrition), they are regularly updated by EFSA11.

In the past, the AR was determined solely by the phenotypic methods, which require the cultivation of the strains tested. Development of molecular methods in recent years has opened also in the area of detection of AR in bacteria the possibilities of much more comprehensive view - either through comparative genomics analysis of whole bacterial genomes, as well as through metagenomic analyses of total bacterial populations from different environments (such as the gastrointestinal tract, food, sewage .. )12–15. In relation with these approaches the term "resistome"16 has been introduced recently which refers to the collection of all AR genes, which are present in a given environment or sample, and can be detected by powerful and high-throughput molecular methods, whichare not limited to the cultivability of bacteria – i.e. the ability to proliferate and grow on the medium.

Significant progress in the research of microbial genomes and microbiomes of different environments requires new approaches also in the research of mechanisms of AR and transmission of AR between bacteria and, more broadly, along the food chain.

Most concerns about the safety of bacteria introduced into food chain through starter cultures, probiotic products and microbial feed additives, are still based on phenotypic antibiotic resistance data of individual strains, not on the whole genome sequencing (WGS) and metagenomic examinations of resistomes of samples from the food chain. The examination of whole genomes of commercial bacteria is recommended in EU, in the latest EFSA-FEEDAP (2018)11 guidelines, for demonstration of the absence of acquired genes coding for or contributing to resistance to clinically-relevant antibiotics, but has not yet been widely implemented in LAB and bifidobacteria. In addition, more comprehensive and global approach is missing, also in the field of probiotics, which are in many ways more controlled as the starter cultures, but the guidelines on the safety assessment of probiotics (FAO/WHO) have not been updated from 2002.

The antibiotic resistance is in EU regularly addressed in the updates of the list of QPS-recommended biological agents intentionally added to food or feed. Phenotypic susceptibility criteria based on minimum inhibitory concentrations (MICs) determination in individual strains, however, are available for a limited number of antibiotics, i.e. 9 for non-enterococcal lactic acid bacteria, 10 for Enterococcus faecium, 8 for bifidobacteria11. Since the commensal bacteria are not studied as extensively as those of clinical relevance, the data on MICs and on the presence of ARG in the commensal bacteria including food industry cultures and probiotics, are not so comprehensive, and therefore the criteria might not be appropriate for all species and antibiotics. 

In the past, the investigation was limited to the phenotypic determination or to the presence of a limited number of the most "risky" genes, which have been demonstrated previously to be involved in horizontal transfer between bacteria. Yet, it should be considered that, even if the individual bacteria do not show phenotypic resistance to certain antibiotics, they may still contain in their genome the genetic elements that are associated with AR. Furthermore, in various environmental samples, not all bacteria are in cultivable state - that is, they cannot be cultivated, as required by the traditional methods for determining of AR. On the other hand, we need to consider that even if transmissible genes are present in commensal bacteria, the actual risk of transmission is poorly known, perhaps even exagerated.

Although microorganisms from the starter cultures used in industrial food production (fermentation and the formation of sensory properties) usually do not survive the passage through the digestive tract as good as probiotics and rarely colonize the intestine, they can nevertheless introduce in this ecosystem DNA which is released from the non-living cells and may contain genetic elements associated with AR. Actually, it is highly likely that we introduce into the body a much larger quantity of starter culture bacteria (viable, non-viable) or their genetic material than we do with probiotics. The most common bacteria present in the starter and probiotic cultures are representatives of the genera Lactobacillus and Lactococcus, in which the AR against certain antibiotics is rather widespread17. Furthermore, also in relation to the trends, which show that the increasing number of probiotics will be marketed in the pharmaceutical field, it is necessary to act quickly.

Determination of bacterial resistomes may assist in the safety assessment of the consumption of lactic acid bacteria and bifidobacteria or products (fermented food, probiotic products ..) containing these bacteria, respectively, and of the potential risk in terms of the spread of AR along the food chain. It also represents a step towards a more prosperous, more in-depth exploration of AR transferability from commensal to pathogenic bacteria within the host (in the gastrointestinal tract of humans or animals).


  • O1: to obtain new information on the presence of genes associated with AR (resistome), in the whole genomes of representatives of starter cultures and probiotic cultures on the basis of already published genome sequences or those obtained in this project by sequencing, and in silico analyses using different bioinformatic tools and databases;
  • O2: to enrich the international database with the MICs of antibiotics important in human and veterinary medicine, for the representatives of lactobacilli and bifidobacteria, which is important for the determination of breakpoint MICs, necessary in the evaluation of the nature of the resistance (acquired or intrinsic) and interpretation of phenotypic test results;
  • O3: to determine the matching of the in silico prediction of ARG and mutations in the whole genomes, and  the results of the phenotypic detection of sensitivity to selected antibiotics;
  • O4: to examine the resistomes of selected fermented and probiotic products (from animal raw materials, containing known starter or probiotic cultures) by metagenomic sequencing, supported by real-time PCR quantification;
  • O5: to asses, on the basis of all results, the burden of probiotic and starter cultures with genes associated with antimicrobial resistance, and to assess the contribution of added starter and probiotic cultures to the resistomes of investigated food samples.


    The main work packages (WP):

    • WP1: Isolation, identification and phenotypic determination of AR,
    • WP2: Sequencing of whole genes and bioinformation analysis,
    • WP3: in silico detection of ARGs and mutations,
    • WP4: Development and validation of selected target ARGs with qPCR,
    • WP5: metagenomic sequencing of food samples and bioinformatic analysis,
    • WP6: synthesis of results and dissemination (writing of articles, conferences).


    Despite the fact that the basic requirements for the safety of all micro-organisms that are intentionally introduced into the food, has been established in EU already in the context of the concept of QPS (2007)10, the market situation shows that there is no adequate control of all products (industrial cultures and final products), containing live microorganisms. Since the legislation on health claims made on foods entered into force, the requirements for probiotic microorganisms, including the risk of AR transmission, are considered more strictly, but we can expect in the near future more strict regulations also in the field of starter cultures for the food industry and end products. Therefore, it is necessary to evaluate the actual situation regarding the prevalence of AR in the segment of starter cultures, food products and probiotics, and develop new, modern approaches to its assessment. The results of the proposed project will help Slovenia to be prepared for tougher requirements on the safety of microorganisms intentionally introduced into the food chain in time, or even a step ahead the other countries.

    The topic of the proposed research contributes to the social goal "Antimicrobial resistance", in particular reducing the transmission of resistant bacteria, in which special attention is paid to further research, which explains how resistance develops in the wider environment, and the development of diagnostic tools and better methods of resistance control. In the EU programs, a lot of attention is given to the problem of the increasing bacterial AR (H2020-EU.3.1 Programme. - Societal CHALLENGES - Health, demographic change and well-being)15. The World Health Organization (WHO) hasalso declared the problem of AR as one of the most important threats to health in the coming period. In the frame of the project, new approaches and methods for the determination of AR (resistome approach, comparative genomics) will be implemented, which is also important from the standpoint of research excellence of participating institutions.


ECDC, Antimicrobial  resistance surveillance in Europe 2015. Annual Report of the European Antimicrobial Resistance Surveillance Network (EARS-Net). Stockholm: ECDC; 2017.

2ECDC/EFSA/EMA. ECDC/EFSA/EMA second joint report on the integrated analysis of the consumption of antimicrobial agents and occurrence of antimicrobial resistance in bacteria from humans and food-producing animals. EFSA Journal. 2017;15:4872.

3Alakomi, H. L. et al. 2016. Quality Assurance and Safety of Crops & Foods, 8, 399 – 413.

4Communication from the Commission to the European Parliament and the Council.

5Strategic research agends – JPIAMR.

6Guidelines for the prudent use of antimicrobials in veterinary medicine, Official journal of the EU, 2015/C 299/04.

7Verraes, C. et al. 2013. International Journal of Environmental Research and Public Health, 10, 2643-2669.

8Rolain, J. M. 2013. Frontiers in Microbiology, 4, 10.

9Gueimonde, M. et al. 2013. Frontiers in Microbiology,

10European Food Safety Authority (EFSA). 2007. Introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. The EFSA Journal, 587, 1-16.

11EFSA FEEDAP. 2018. Guidance on the characterisation of microorganisms used as feed additives or as production organisms. EFSA Journal 16(3):5206, 24 pp.   

12Penders, J. et al. 2013. Frontiers in Microbiology, 4.

13Pärnänen, K. et al. 2018. Nature Communications, 9, 10.1038/s41467-018-06393-w

14Duranti, S et al. 2017. Applied and Environmental Microbiology, 83. UNSP e02894-16.  

15Rodríguez, C. et al. 2019.

16Wright, G. D. 2010. The antibiotic resistome. Expert Opinion on Drug Discovery, 5, 779-788.

17Devirgiliis, C., Zinno, P. & Perozzi, G. 2013. Frontiers in Microbiology, 4, 13.