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“Mobile genetic elements mapping – first bioinformatic insight into the resistome of ESKAPE clinical and environmental isolates from Romania”

Project Title:Mobile genetic elements mapping – first bioinformatic insight into the resistome of ESKAPE clinical and environmental isolates from Romania

Project Number: PN-III-P1-1.1-PD-2021-0540

Funding Agency: UEFISCDI

Project Leader: Marius Surleac

Mentor: Carmen Mariana Chifiriuc

Acronym: EMPIRE

Project Duration: 2022 – 2024

Project Description:

Among European countries, Romania is experiencing one of the highest rates of antibiotic resistance (AR) as revealed by most recent data reported by international surveillance programs such as ECDC, justifying the urgent need for monitoring the AR dynamics in the clinical and environment isolates. This project aims to contribute to the study of mobile genetic elements (MGEs) which are important contributors to the worldwide dynamics of resistance spreading. This project attempts to characterize in detail the ESKAPE resistome by bioinformatic analyses of various MGEs (e.g., plasmids, insertion sequences, transposons, genomic islands) representing huge pools of resistance genes and sets of such genes. These aspects will be correlated with bioinformatic studies focused on the phylogenetic relationships between such factors, between the isolates from the same species and among species, which in turn will cover some vital aspects of clinical interest, namely, functional traits with impact on the metabolic pathways, biological processes. Another important objective of this proposal is to validate the bioinformatic analysis by the use of experimental methods such as the MinION (Oxford Nanopore) technology for 3rd generation sequencing; this will improve the results by increasing the coverage and the degree of confidence: by using such a method we’ll be able to capture the full length of genes with correct orientation as well as of big MGEs such as genomic islands and plasmids.

Objectives:

2022

A1. Obtaining the bacterial mobile genetic elements’ reference sequences.

A2. New bioinformatic approaches based on NGS sequencing data – Annotation of the assembled Romanian genomes and the pangenome analysis – Part I.

2023

A3. New bioinformatic approaches based on NGS sequencing data – Adnotation of the assembled Romanian genomes and the pangenome analysis – Part II.

A4. Functional analysis.

A5. Mobile Genetic Elements (MGE) predictions – Part I.

A6. Experimental validation by 3rd-generation sequencing analysis of AMR genes transported by plasmids or other MGEs.

Results – 2022:

Scientific papers:

Gheorghe-Barbu, I., Barbu, I.C., Popa, L.I. et al. “Temporo-spatial variations in resistance determinants and clonality of Acinetobacter baumannii and Pseudomonas aeruginosa strains from Romanian hospitals and wastewaters”, Antimicrob Resist Infect Control, 11, 115 (2022). PubMed [36104761]. (Equal contribution – Main Author).
IF: 5.5; AI: 1.382

Oral and Poster presentations:

An Overview On The Dynamics Of Mobile Genetic Elements In Clinical And Environmental WGS ESKAPEE Pathogens In Romania” (Marius Surleac, Ilda Czobor Barbu, Simona Paraschiv, Irina Gheorghe, Robert Hihan, Carmen Chifiriuc, Dan Oțelea) – 6th Conference on the Environmental Dimension of Antibiotic Resistance (EDAR6), Gothenburg, Sweden, September 22-27, 2022. (Poster Presentation P1-009)

Antibiotic And Biocides Resistance Genes In Acinetobacter Baumannii Clinical And Wastewater Strains Before And After The Onset Of COVID-19 Pandemic” (Ilda Czobor Barbu, Irina Gheorghe, Marius Surleac, Simona Paraschiv, Gratiela Gradisteanu Pircalabioru, Dan Otelea, Mariana Carmen Chifiriuc) – 6th Conference on the Environmental Dimension of Antibiotic Resistance (EDAR6), Gothenburg, Sweden, September 22-27, 2022. (Poster Presentation P1-085)

Wastewater treatment plants at the crossroads of the aquatic and clinical reservoirs of antimicrobial resistance – The radar study” (Mariana Carmen Chifiriuc, Marcela Popa, Luminita Marutescu, Ilda Czobor, Irina Gheorghe, Gratiela Gradisteanu, Dan Otelea, Simona Paraschiv, Marius Surleac, Mircea Ioan Popa, Cerasela Dragomirescu, Alexandru Muntean, Codruta Usein, Mihaela Oprea, Sorin Dinu, Mihai Nita Lazar, Anton Ficai) – Symposium of International Chemical Engineering and Materials (SICHEM), UPB, Bucharest, Romania, 17 – 18 November, 2022. (Oral Presentation)

Turning the Escherichia coli upside-down – an overview on the pathogenicity and MGE of strains circulating in Romania between 2019-2020” (Marius Surleac, Ilda Czobor Barbu, Simona Paraschiv, Irina Gheorghe-Barbu, Robert Hohan, Carmen Chifiriuc, Dan Otelea) – 18th Edition of The Scientific Days of the National Institute for Infectious Diseases „Prof. Dr. Matei Balș” National Congress, Sinaia, Romania, November 21-24, 2022. (Oral Presentation)

Results – 2023:

Scientific papers:

Stanciu AM, Florea D, Surleac M*, Paraschiv S, Oțelea D, Tălăpan D, Popescu GA. “First report of Candida auris in Romania: clinical and molecular aspects”, Antimicrob Resist Infect Control, 12(1):91 (2023). PubMed [37674189]. (Equal contribution – Main Author).
IF: 5.5; AI: 1.382

Oral and poster presentations:

Antimicrobial resistance, MGEs and functional profiling through use of shotgun metagenomics on isolates from receiving surface waters proximal to WWTPs” (M. Surleac*, I. Czobor Barbu, S. Paraschiv, I. Gheorghe-Barbu, D. Oțelea, M.C. Chifiriuc), „The dynamic changes of human microbiome after fecal transplantation – case report” (Marius Surleac, Simona Paraschiv*, Leontina Banica, Catalin Apostolescu, Dragos Florea, Dan Otelea) – 8th International Conference on Clinical Metagenomics; & Cell Press LabLinks meeting ‘Antimicrobial resistance: from mechanisms to global implications’, 15-17 November 2023, Geneva, Switzerland. (Poster Presentations: P-18 & P-19).

Bioinformatic Insights into Resistant Escherichia Coli Isolated from Different Aquatic Environments in Romania” – (M. Surleac, I. Czobor Barbu, S. Paraschiv, I. Gheorghe-Barbu, D. Oțelea, M.C. Chifiriuc) &Comparative Phenotypic and Molecular Characterization of Clinical and Aquatic Multidrug Resistant Acinetobacter Baumannii Circulating Clones Isolated in Romania for Four Consecutive Years” (I. Gheorghe-Barbu, I. Czobor Barbu, V.M. Corbu, G.A. Grigore, O.C. Vrâncianu, M. Surleac, S. Paraschiv, L. Bănică, A.M. Muntean, I. Pecete, L. Măruțescu, M. Popa, M.I. Popa, D. Oțelea, M.C. Chifiriuc) – Session 5 (Biologically active substances, transformation products and antibiotic resistance determinants in wastewater and sludge receiving environments), 18th International Conference on Chemistry and the Environment (ICCE 2023), 11 – 15 June, 2023, Venice, Italy. (Oral Presentations: OP-404 & OP-225).

Antibiotic resistance shuttles in Romanian ESKAPE isolates from surface/used waters and clinical isolates” – (Marius Surleac, Ilda Czobor Barbu, Simona Paraschiv, Irina Gheorghe-Barbu, Robert Hohan, Liviu I Rotaru, Carmen Chifiriuc, Dan Otelea); 2023 RoBioinfo Conference, 11th-13th May 2023, Bucharest, Romania. (Oral Presentation).

Participation in the course:

Participation in „How to design and deliver pathogen genomics training for health and research professionals”, Wellcome Genome Campus, 26-29 Iunie 2023, Hinxton, Cambridge, UK. The course has been organised by Wellcome Connecting Science & Centre for Genomic Pathogen Surveillance. (The selection and acceptance was based on CV)

Workshop (facilitator):

Hands-on Pathogen genomics course – from sequence analysis to impact on public health” – 23-24 Octombrie 2023, Cluj Napoca, Romania. The workshop was conducted together with Monica Abrudan (CGPS, University of Oxford, UK) and organised by the Romanian Society of Bioinformatics & the Faculty of Biology and Geology at the ”Babeș-Bolyai” University.

Results – 2024:

Scientific papers:

Irina Gheorghe-Barbu, Marius Surleac, Ilda Czobor Barbu, Simona Paraschiv, Leontina Mirela Bănică, Liviu-Iulian Rotaru, Ovidiu Corneliu Vrâncianu, Mihai Niță Lazăr, Dan Oțelea, Mariana Carmen Chifiriuc, ”Decoding the resistome, virulome and mobilome of clinical versus aquatic Acinetobacter baumannii in Southern Romania” (under evaluation at Science of the Total Environment). (Equal contribution – Main Author).

Oral and poster presentations:

Clustering in Context: Antimicrobial Resistance and MGE Dynamics in ESKAPE Pathogens Circulating across Environmental and Used WWTP Waters in Romanian Cities” (M. Surleac*, I. Czobor Barbu, S. Paraschiv, I. Gheorghe-Barbu, Liviu I. Rotaru, D. Oțelea, M.C. Chifiriuc) – 7th Environmental Dimension of Antimicrobial Resistance conference (EDAR7), May 26-31, 2024 in Montreal, Quebec, Canada. (Oral Presentation: 399).

Bioinformatic Silver nanoparticle as therapeutic options to fight the resistome, mobilome and virulome of multidrug resistant Acinetobacter baumannii circulating clones in South Romania” (I. Gheorghe-Barbu, I. Czobor Barbu, V.M. Corbu, A-Ș. Dumbravă, D-M Găboreanu, G.-A Grigore, C.O. Vrâncianu, M. Surleac, S. Paraschiv, D. Oțelea, D. Ficai, A. Ficai and M.C. Chifiriuc) – Polymers 2024, Composites 2024 and 3BS Materials 2024 International Joint Conference, 06 – 08 March 2024, Seville, Spain. (Poster Presentation).

***

Scientific Report – Part I
The first stage of this project proposes the following: i) obtaining the reference sequences of the mobile genetic elements (MGE) (e.g. insertion sequences, transposons, integrons, genomic islands, etc.) but also other antibiotic & biocide resistance and virulence determinants, ii) adnotation of over 700 sequences (corresponding to multiresistant strains from ESKAPEE group, isolated from different geographical regions from Romania, having the complete genomes sequenced in our lab), iii) the pangenome analysis of these strains. The MGE sequences have been downloaded from various databases and then organised by various categories, in order to be compared with the sequencing data of the Romanian isolates. Therefore, a set of preliminary analyses has been performed, underlining a high diversity of circulating MGEs in Romania, which will further be analyzed in more detail through the next stages of this project. The adnotations, the pangenome and phylogenetic analyses of the strains belonging to various ESKAPEE pathogen species from the analyzed set, suggest that these pangenomes are open, that they have a set of common genes for all the strains in the same species, but also accessory genes that could show genomic signatures specific to a geographical region or to clinical origins and aquatic environments as well. All these results, together with the ones that will be analyzed in the next stage of the project will provide useful information for a better understanding of the resistome dynamics in the ESKAPEE strains circulating throughout Romania.

Scientific Report – Part II
The second stage of this project proposes the following: i) annotating the obtained Romanian genomic sequences, and the pangenome analysis; ii) the functional analysis of the sequenced strains; iii) mobile genetic elements (MGE) predictions; iv) experimental validation (by 3rd-generation sequencings methods) of the ARGs carried by plasmids or other MGEs. The final annotation, pangenome and phylogenetic analyses, of the whole set of sequenced strains show that the obtained pangenomes for each species are “opened” pangenomes, suggesting increased dynamics of the species circulating in various environments allover Romania. This observation is backed also by the results of functional analysis on these strains, and by the results that we have on a small set of shotgun metagenomic sequencing data, as well. Putting all these results together it helps us building the foundation of a One Health approach in Romania, hoping at the extent of such studies in the near future. Aside of that, the bioinformatic analysis methods that were started with this project, will soon be used by the scientific community.

Scientific Report – Final Report
Overall, all the objective in this research grant have been fulfilled and described in detail in the annual scientific reports but also in the final report. All the obtained results have been, so far, published as two scientific papers and presented at ten national and international conferences, and will further be published as such in the future. The huge data we have so far could provide useful information for a better understanding of the dynamics and mechanisms behind the resistome of ESKAPEE species, but also in other species in Romania. Probably the most significant result is represented by the development of complex analysis methods but also as complete as possible through the use of bioinformatic instruments, such a complete approach is not implemented in general in the available online tools. This aspect together with the over 900 analysed pathogen isolates gives us an idea, a more detailed image on the resistance and transmission mechanisms, starting an One Health approach in Romania, hopeing that in the near future, such studies will increase in numbers with the sole purpose of surveilling not only the clinical but also the environment and the animals within. More than that, these bioinformatic analysis methods developed throughout this project will be available soon to be used by the Romanian and international scientific community. Last but not least, running research grant in good conditions made possible the collaboration with other research groups abroad, collaborations that are ongoing even after the end of this research project, and hopefully will be a starting point for other research grants.

***

Marius Surleac, PhD – Links & Highlights: Hirsch = 12 (Google Scholar); Cumulative Impact Factor = 136.735; Cumulative AIS = 48.57; Google Scholar; PubMed.

Carmen Mariana Chifiriuc, Prof., PhD – Links & Highlights: Hirsch = 50 (Google Scholar); Google Scholar; Pubmed.

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