L.N. Gumilyov Eurasian National University, a dissertation defense for the degree of Doctor of Philosophy (PhD) by Bayanbek Dina on the topic «Multi-omics approach to studying the cellular response of Bacteroides fragilis to carbapenem» to the educational program «8D05107 – Biology».
The dissertation was carried out at the «General Biology and Genomics education department» of L.N. Gumilyov Eurasian National University.
The language of defense is kazakh
Official reviewers:
Yernazarova Aliya Kulahmetovna - Candidate of Biological Sciences, Leading Researcher at the Research Institute for Environmental Problems of Al-Farabi Kazakh National University (Almaty, Kazakhstan).
Tynybayeva Indira Kazhymukhanovna - Candidate of Biological Sciences, PhD, Senior Researcher at Republican Collection of Microorganisms LLP (Astana, Kazakhstan).
Temporary members of the Dissertation Council:
Sarmurzina Zinigul Serikovna - Candidate of Biological Sciences, Associate Professor, Head of the Biotechnology Laboratory at Republican Collection of Microorganisms LLP (Astana, Kazakhstan).
Sandybaev Nurlan Tamambaevich - Candidate of Biological Sciences, Professor, Director of the Kazakhstan–Japanese Innovation Center at the Kazakh National Agrarian Research University (Almaty, Kazakhstan).
Muhammad Qasim - PhD, Professor, Department of Microbiology, Kohat University of Science and Technology (Kohat, Pakistan).
Scientific advisors:
Ilderbaev Oralbek Zainuldanovich – Doctor of medical sciences, Professor of the Department of «General biology and genomics», L.N. Gumilyov Eurasian National University, Kazakhstan.
Zholdybaeva Elena Vitalyevna – Candidate of Biological Sciences, Professor, Head of the Laboratory of «National Scientific shared Laboratory of Biotechnology», National Center for Biotechnology, Kazakhstan.
Soki Jozsef – PhD, Senior Research Fellow, Institute of Medical Microbiology, University of Szeged, Szeged, Hungary.
The defense will take place on June 12, 2026, at 10:00 AM in the Dissertation Council for the training direction «8D051 – Biological and related sciences» under the educational program «8D05107 – Biology» of L.N. Gumilyov Eurasian National University. The dissertation council meeting will be held in a mixed offline and online format.
Link: https://us06web.zoom.us/j/89115621409?pwd=B5Gh1bGxfsHwEbgfB0kBBvfdqnvaFa.1
Address: Astana, Kazhymuqana str., 13, building №310
Abstract (English): General description of the work The dissertation is devoted to a multi-omic approach to studying the cellular response of Bacteroides fragilis to one of the most active antimicrobial drugs against B. fragilis, meropenem. Studying the transcriptome and proteome of the bacterium will help us understand how bacterial genes associated with resistance mechanisms and metabolic pathways involved in the response to carbapenems are related to the frequency of meropenem. Relevance of the study Antimicrobial resistance remains a significant global threat to public health. In 2019, drug-resistant infections caused more than four million deaths worldwide. Projections show that by 2050, antimicrobial resistance could cause up to 10 million deaths annually and cost the global economy more than $100 trillion. Consequently, there is an urgent need for strategies to combat antimicrobial resistance. Today, the prevalence of antibiotic resistance in clinical strains of B. fragilis is a pressing issue worldwide. Studies conducted in Europe have shown that over the past 20 years, there has been an increase in imipenem resistance in B. fragilis from 0% to 1.2% in European countries, with a similar increase in imipenem resistance also observed in the United States. Studies conducted in Taiwan between 2008 and 2012 also showed that 13.5% of B. fragilis isolates are resistant to ertapenem. In addition, there are reports of multidrug resistance of B. fragilis strains to antibiotics. In 2022, 449 unique B. fragilis isolates isolated from blood were registered as sensitive to antimicrobial drugs in 16 European countries and resistance to meropenem (13.4% – 45.5%). Resistance to meropenem also increased from 3.4% to 10.7%. The development of high-throughput and high-tech innovative methods in biology has contributed to the development of “omic” technologies. In recent years, the multi-omic approach has been integrated into many areas of biology, particularly microbiology. The study of protein profiles (proteomes) of bacterial pathogenesis is one of the main approaches to the study of proteins and pathogen-host interactions with the aim of gaining a deeper understanding of the mechanisms of bacterial resistance and virulence. Chernov A. and his colleagues showed in their review the relevance of using “omic” technologies to understand the mechanisms of bacterial resistance and the use of the data obtained to create new antimicrobial drugs. For example, there are quite a few studies on Mycobacterium tuberculosis and Salmonella based on the multi-omic approach. One of the key drugs used in antimicrobial therapy against B. fragilis are carbapenems. Resistance to carbapenems in B. fragilis is associated with the production of a metal-β-lactamase with two Zn2+ ions in the active site, encoded by the cfiA gene, which is sometimes silent. An increase in the frequency of isolation of Bacteroides spp. strains resistant to carbapenems is preceded by an increase in the carriage of the cfiA gene. Resistance to carbapenems is due to the expression of the cfiA gene, which is regulated by mobile genetic IS elements. For example, IS1186 and IS942 are well-known mobile genetic elements involved in regulation. Sóki J et al. also report on previously unknown mobile IS elements, IS943, and the need for more detailed study of the regulation of cfiA gene expression. In a study by Wafaa Jamal et al. on the prevalence of resistance and sensitivity of B. fragilis isolates in Kuwait, it was noted that one of the B. fragilis isolates was resistant to both imipenem and meropenem, although the cfiA gene was absent. The authors attribute this resistance to another mechanism—impaired membrane permeability. In addition, a relatively recent study by Pak-Leung Ho et al. showed that the acquisition of resistance to imipenem in cfiA-positive B. fragilis may be mediated not only by the expression of the cfiA gene due to IS insertion, but also by other undefined mechanisms. Potential mechanisms of resistance may include mutations leading to porin loss, increased drug efflux, or decreased affinity of penicillin-binding proteins. Purpose and objectives of the study The purpose of the study is to evaluate in vitro the transcriptional and proteomic profile of B. fragilis when exposed to meropenem in order to study the relationship between bacterial genes and resistance mechanisms and metabolic pathways involved in the response to meropenem. 1. Cultivation of B. fragilis isolates. Determination of the subinhibitory concentration (SIC) of meropenem. Subcultivation of B. fragilis isolates in the presence and absence of meropenem antibiotic at different time intervals. 2. Determination of bacterial RNA profiles of B. fragilis under the influence of an antibiotic (meropenem) and upon removal of this antibiotic. The essence of the task is to compare the whole-genome gene expression profile of B. fragilis when exposed to a subinhibitory concentration of the antibiotic after different periods of time. RNA sequencing (RNA-seq) will be performed using NGS (next-generation sequencing). 3. Determination of the protein profile of B. fragilis when exposed to the antibiotic (meropenem) and when this antibiotic is removed. The essence of the task is to quantitatively determine the regulation of B. fragilis proteins when exposed to a subinhibitory concentration of the antibiotic after different periods of time. A one-dimensional gel electrophoresis method will be used in combination with chemical labels to detect changes in the regulation and chemical modification of individual proteins within the entire proteome in response to the antimicrobial drug meropenem. 4. Conducting bioinformatic analysis of B. fragilis transcriptome and proteome data. The essence of the task is to identify the main activated or repressed genes encoding enzymes that participate in the response to meropenem, as well as to identify key proteins of B. fragilis that are part of the cellular response activated by the antimicrobial drug. Research object The research object was the B. fragilis BFR KZ01 culture, which was isolated in 2018 at Astana city hospital No. 1 from a 48-year-old patient diagnosed with acute gangrenous perforative appendicitis and peritonitis. Research methods The research used modern microbiological and molecular biological methods – cultivation, RNA isolation, next-generation sequencing, and mass spectrometric analysis of proteins. Scientific novelty of the research The dissertation is aimed at obtaining new data on the mechanisms of B. fragilis resistance to carbapenems. Prior to this study, no research on bacteria based on a multi-omic approach had been conducted in Kazakhstan. Practical significance Based on the results of the dissertation, it has been shown that low doses of antibiotics activate protective mechanisms that contribute to the development of resistance. The data obtained can subsequently be used to create targeted antimicrobial drugs. Main conclusions based on the research results The following scientific results are presented for defense: 1. The subinhibitory concentration of the antibiotic meropenem for the clinical strain B. fragilis BFR KZ01, which ensures the development of resistance, has been determined. The minimum inhibitory concentration (MIC) was determined to be 1 μg/ml, and the SIC of meropenem was 0.5 μg/ml (0.5 MIC). 2. B. fragilis BFR KZ01. The raw RNA profile data for B. fragilis BFR KZ01 - SRA is deposited in NCBI under no. SRX22081155. Mapping of reads to the reference genome revealed 2,477 expressed genes among all B. fragilis BFR_KZ01 samples. When comparing wMEM (parent strain) and under the influence of the antibiotic meropenem (FDR < 0.05), 82 differentially expressed genes were found, of which 47 genes were overexpressed and 35 were underexpressed; When comparing MEM2 with rMEM8, 49 were up-regulated and 46 were down-regulated. When comparing wMEM with rMEM8, 45 were up-regulated and 33 were down-regulated. 3. The proteomic profile of the clinical strain B. fragilis BFR KZ01 was determined. A total of 859 proteins of the clinical strain B. fragilis BFR KZ01 were identified, and 108 differentially expressed proteins were identified using filtration steps. 4. Ten DEGs were common to the comparison groups during and after antibiotic withdrawal (wMEM vs MEM2 and MEM2 vs rMEM8). Cluster analysis revealed a statistically significant enrichment cluster (W-0560 oxidoreductase) of DEGs after antibiotic withdrawal. The activation of approximately 7 genes associated with molecular function and transmembrane transport activity was noted, which once again highlights the main mechanism of resistance, involving the active exclusion of antimicrobial drugs from cells by drug transport systems. Three statistically significant protein-expressing genes were identified: 3-oxoacyl-[acyl-carrier protein] reductase, biotin carboxylase subunit acetyl-CoA carboxylase, and beta-ketoacyl-APB synthase III. These proteins are necessary for the proper initiation and elongation of fatty acid chains during the formation of membrane lipids and various cellular processes in B. fragilis. Based on the research results, six papers were published, including one article in a journal listed in the Scopus database (Heliyon, IF 3.4, percentile 82), three articles in journals recommended by the Committee for Quality Assurance in Education and Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan, and two articles in collections of international scientific conferences. Structure of the dissertation The dissertation is presented on 92 pages, 16 tables, 36 figures and 8 appendices. The work consists of an introduction, a review of the literature, research materials and methods, results and discussion, conclusions, a list of sources used, including 187 sources and appendices.
Conclusion of the Research Ethics Committee
Defense of the dissertation: https://youtu.be/2BMf5yKTUJI?si=uK5LpdJBfVOhSirs
