top of page

Microbiological diagnostics: From traditional to molecular genetic methods: A literature review


ree


Підписуйтесь на наші соціальні мережі, щоб стежити за останніми новинами тут 💜:

Сайт: www.ediens.me


Article’s History: Received: 02.08.2023; Revised: 16.10.2023; Accepted: 28.11.2023

DOI: 10.61751/bmbr/4.2023.34 Journal homepage: https://bmbr.com.ua/en

UDC 579-043.37+ 577.21

Microbiological diagnostics:

From traditional to molecular genetic methods: A literature review

Maxim Ivashko

Postgraduate Student, Assistant Professor

Uzhhorod National University

88000, 3 Narodna Sq., Uzhhorod, Ukraine

Svitlana Burmei*

Postgraduate Student, Assistant Professor

Uzhhorod National University

88000, 3 Narodna Sq., Uzhhorod, Ukraine

Lesya Yusko

PhD in Biological Sciences, Associate Professor

Uzhhorod National University

88000, 3 Narodna Sq., Uzhhorod, Ukraine

Tetiana Chaikovska

PhD in Medical Sciences, Associate Professor

Uzhhorod National University

88000, 3 Narodna Sq., Uzhhorod, Ukraine

Nadiya Boyko

Doctor of Biological Sciences, Professor

Uzhhorod National University

88000, 3 Narodna Sq., Uzhhorod, Ukraine



Abstract. The development of new and optimisation of known cultural and molecular genetic methods for accurate

species identification of microorganisms is an urgent and practically necessary task that is receiving great attention

from researchers. The purpose of this study was to analyse and systematise theoretical scientific data on methods of

microbial identification and assess their main advantages and disadvantages. For this purpose, a systematic review of

53 randomised research papers published between 2018 and 2023 was conducted. The search for publications using the

keywords “microbiome”, “microbiological diagnostics”, “identification of microorganisms”, “sequencing”, and “omics

technologies” in the title or text of the research paper was carried out in Web of Science, Scopus, PubMed, and Google

Scholar. The study provides generalised information on traditional and modern methods of microbial identification. It

is established that the advantages of traditional diagnostic methods include the possibility of preserving the obtained

microorganisms for further research, in particular, for determining their antibiotic sensitivity. Modern molecular

genetic methods open up new possibilities for the accurate identification of microorganisms, including those that are

difficult to incubate using traditional cultural methods. The use of these methods allows obtaining detailed data on the

genetic structure and diversity of microorganisms, which is important in many fields, including microbiology, medicine,

and ecology. However, molecular methods are more sensitive to contamination or errors in the sample collection

and processing. Moreover, omics technologies (genomics, transcriptomics, proteomics, and metabolomics) open up

new opportunities for studying cells at more complex levels of life organisation. In everyday clinical practice, the

microbiological method of isolating microbial cultures remains the main one, as it is the only one that allows determining

not only the cause of the infection but also the antibiotic sensitivity. However, cultural methods for studying fastidious

microorganisms are quite limited. Thus, the results obtained contribute to the development of fast and accurate

methods for identifying, classifying, and systematising microorganisms, which facilitates the processes of diagnosis and

development of strategies for the control and treatment of infectious diseases in laboratories and clinical institutions

Keywords: microbiology; diagnostics; nutrient medium; identification; sequencing; omics technologies.


e INTRODUCTION Microorganisms can affect a person’s life both positively and negatively. People have learned to use microorganisms in various areas of their lives. They are closely related to medicine, biotechnology, nutrition, genetic engineering, etc. As noted by Y. Gao et al. [1], the ability of bacteria and various types of fungi to produce antimicrobial substances makes them potentially useful in drug production. From the earliest childhood, the human body is also inhabited by a diverse community of microorganisms, which consists of  bacteria, fungi, viruses, archaea, and is called the microbi- ome [2]. The human microbiome is a collection of microbi- ocenoses that colonise all surfaces of the human body, in- cluding the skin, respiratory system, gastrointestinal tract,  and genitourinary system. These microorganisms perform  a number of functions necessary for maintaining the ho- meostasis of the human body [3].  However, certain genetic features of bacteria make  them dangerous to human health [4]. Antimicrobial re- sistance, which develops as a result of uncontrolled use  of antibiotics, excessive use in animal husbandry and the  food industry, poor infection control in hospitals and clin- ics, and lack of hygiene [5], are recognised as serious in- ternational problems. This problem makes it impossible to  successfully treat many common bacterial infections and causes a growing number of deaths. A. Salmanov et al. [6]  suggested possible ways to develop a multidisciplinary ap- proach based on the principles of the “unified health” con- cept to solve problems related to antibiotic resistance.  Effective treatment becomes difficult without labo- ratory diagnostics, as the lack of diagnostic tests makes  it difficult to establish any diagnosis. It is the qualitative diagnosis that determines the correct decisions of the doctor regarding the treatment methods used, evaluating  their effectiveness, and in many cases preventing the oc- currence of the disease due to the detection of pathology  at an early stage of development [7]. Rapid identification of microorganisms plays a key role in the fight against infections. This helps to accurately identify the types of microorganisms that cause diseases and determine their sensitivity to antimicrobials. However, without rapid and accurate identification of infectious agents, complications associated with the wrong choice of antibiotic therapy for the patient may occur.  The range of diagnostic approaches used in microbiol- ogy is extremely wide and ranges from traditional to molec- ular genetic methods. Each method has its own strengths  genetic methods open up new possibilities for the accurate identification of microorganisms, including those that are difficult to incubate using traditional cultural methods. The use of these methods allows obtaining detailed data on the genetic structure and diversity of microorganisms, which is important in many fields, including microbiology, medicine, and ecology. However, molecular methods are more sensitive to contamination or errors in the sample collection and processing. Moreover, omics technologies (genomics, transcriptomics, proteomics, and metabolomics) open up new opportunities for studying cells at more complex levels of life organisation. In everyday clinical practice, the microbiological method of isolating microbial cultures remains the main one, as it is the only one that allows determining not only the cause of the infection but also the antibiotic sensitivity. However, cultural methods for studying fastidious microorganisms are quite limited. Thus, the results obtained contribute to the development of fast and accurate methods for identifying, classifying, and systematising microorganisms, which facilitates the processes of diagnosis and development of strategies for the control and treatment of infectious diseases in laboratories and clinical institutions Keywords: microbiology; diagnostics; nutrient medium; identification; sequencing; omics technologies  Microbiological diagnostics...  36  Bulletin of Medical and Biological Research. 2023. Vol.5, No. 4 For this purpose, a systematic review of 53 randomised research papers published between 2018 and 2023 was  carried out. The criteria for inclusion in the study includ- ed scientific publications in Ukrainian and English. The  process of selecting publications included the stages of screening and quality assessment, where each paper was carefully analysed with regard to its scientific content and methodological aspects. Using electronic databases such as Web of Science, Scopus, PubMed, and Google Scholar, the authors searched for publications using the keywords “microbiome”, “microbiological diagnostics”, “microbial identification”, “sequencing”, and “omics technologies” in the title or text of the paper. It is important to note that the included papers were analysed according to various  criteria, such as the year of publication and the main re- sults. This approach helps to systematically summarise the  literature data and provides a deeper look at the selected publications for the study. e TRADITIONAL METHODS OF MICROBIAL IDENTIFICATION  Most methods of isolation and identification of micro- organisms remain unchanged and are based on the use  of specific nutrient media that allow studying their vari- ous properties. Microorganisms differ in their needs, and  therefore, there is no single environment or set of condi- tions that would ensure the growth of all species.  Conventionally, Nutrient agar, a universal medium, is  used for the cultivation of microorganisms. By their com- position, such nutrient media are designed for the culti- vation of a wide range of microorganisms. Preparation of  blood agar (BA) by adding 5% sheep blood to Nutrient agar expands the possibilities for studying and culturing those microorganisms that may require specific components or  conditions for growth [13]. Thus, by the nature of haemol- ysis on BA, primary identification of streptococci is carried  out. To identify certain infectious agents, it is important to use more specific nutrient media. For example, differential diagnostic nutrient media allow determining differences in the metabolic activity of microorganisms using systems  of biochemical indicators. Nutrient media are also divid- ed into selective ones, which are characterised by selective  growth of a certain type of microorganism due to the addi- tion of antibiotics to the nutrient medium. Metronidazole  is often added to the nutrient medium for selective isola- tion of anaerobic microorganisms.  Chromogenic media are widely used in microbiological practice to identify specific types of microorganisms. The  fluorogenic substrates present in these media are hydro- lysed under the influence of specific enzymes produced by  each type of microorganism. Hydrolysis of the fluorogenic substrate can lead to a change in the colour of the medium, which allows rapid and efficient identification of species [14].  After isolation of a pure culture, the phenotypic prop- erties of the microorganism under study are determined for  further identification. One of the main tests is the Gram stain method, which distinguishes between gram-positive  and gram-negative microorganisms. The basic biochemi- cal tests in clinical microbiology are the determination of  catalase, oxidase, indole H2  S, etc. Most modern biochemi- cal test kits include a combination of a wide range of bio- chemical determinations that are combined in a single test  panel. This facilitates and speeds up the microbial identifi- cation process, providing faster and more accurate results.  Although traditional methods are available and pro- vide information on the diversity of microorganisms in  the sample, both quantitatively and qualitatively, the time required for identification based on traditional methods is estimated at a minimum of 2 to 5 days [15]. In addition,  traditional methods do not provide an opportunity to iden- tify uncultivated microorganisms. However, the advantag- es of this method are the ability to preserve the resulting  microorganisms and test their sensitivity to antibiotics for effective treatment of the patient.  Immunological methods. Such immunological diagnos- tic methods in microbiology as agglutination, serology,  immunoblotting, enzyme-linked immunosorbent assay (ELISA), etc., are worthy of attention. The basic principle of immunological methods is to investigate the interaction between an antibody and an antigen. These methods help in the identification of various microorganisms [16], but their use is accompanied by a number of limitations and  disadvantages. Despite the high specificity of the analy- sis, this can lead to incorrect results or underestimate the  presence of a pathogen. Some immunological methods are  expensive, which can be a limiting factor for many labora- tories due to the use of expensive equipment and reagents.  In addition, the presence of chronic or immunodeficiency conditions can affect the accuracy and reliability of this method [17]. The unfavourable aspects of these methods do not reduce their importance as tools for studying and diagnosing microorganisms. Properly used immunological methods remain key in determining the immune response and identifying pathogens. Mass spectrometry. Improvements in the identification of microorganisms have arisen from the need to reduce the  time, cost, and simplify the procedures required to accu- rately identify these microorganisms. Therefore, the rap- id development of mass spectrometry (MS) has led to its  widespread use in microbiology [18]. The high speed, re- duced cost, and ease of use of MS greatly facilitated and  accelerated the identification of microorganisms compared to traditional cultural methods. The main principle of MS  is the analysis of ions based on their mass-to-charge ra- tio (m/c), which allows studying the molecular structure,  mass, and concentration of substances in various types of samples [19]. The combination of MS with gas or liquid chromatography significantly expanded the possibilities of this powerful method [20]. This has contributed to a better understanding of biological systems, allowing the analysis of a wide range of biomolecules.  The basic principle of gas chromatography mass spec- trometry (GC-MS) is that first, using gas chromatography,  the substances of the test sample are separated according to their properties. Next, the separated components are  analysed in a mass spectrometer, where individual com- ponents are identified by their chemical properties [19].  This method is usually applied to identify microorganisms based on their lipid structures. Thus, S.P. Putri et al. [21] investigated 11 undetectable pathogenic Corynebacteria  using GC-MS based on mycolic acid analysis. The effec- tiveness of the GC-MS method has also been demonstrated  on yeast-like fungi [22]. The liquid chromatography mass spectrometry (LC-MS) method is based on the separation  M. Ivashko et al.  37  Bulletin of Medical and Biological Research. 2023. Vol.5, No. 4  of substances in a sample using the liquid phase and the  analysis of the mass and charge of ions in a mass spectrom- eter. At this stage, individual components are identified by  their mass and chemical properties [20].  Matrix-assisted laser desorption ionisation time-of- flight mass spectrometry (MALDI-TOF MS) is an advanced  tool for the rapid and accurate identification and classi- fication of bacteria [23], fungi [24], and viruses [25]. The  principle of MALDI-TOF MS is to ionise microorganisms using laser radiation, which leads to the formation of ions.  These ions are accelerated in a vacuum system by an elec- tric field and move towards the mass spectrometer detec- tor in a certain way. The detector registers their mass, and  the obtained data are used to identify microorganisms [26]. Among other applications, MALDI-TOF MS is also used to identify various metabolites. For example, the study by  J. Doellinger et al. [27], using MALDI-TOF MS, investigat- ed enterotoxins produced by pathogenic strains of Bacil- lus cereus. MALDI-TOF MS is also used to quickly detect  antibiotic resistance genes in bacteria [28], which allows improving treatment strategies for infections, especially those caused by polyresistant strains of microorganisms. Bacterial identification using MALDI-TOF MS mainly focuses on improving methods for isolating and cleaning  pathogens from clinical samples, expanding spectral li- braries and updating software. With the development of  technology, many MALDI-TOF MS-based microbial iden- tification databases and systems have been licensed and  put into clinical use. However, there is a need to further improve the antimicrobial resistance analysis based on  MALDI-TOF MS to provide comprehensive clinical micro- biological characterisation [29]. The application of MAL- DI-TOF MS for the analysis of small molecules is usually  limited by the choice of a suitable matrix. Matrices used for  large-molecule analysis are often not suitable for analys- ing low-molecular-weight compounds (m/c <1,000 Da) due  to matrix background noise, ion suppression, and uneven crystallisation by traditional organic matrices. e MOLECULAR GENETIC METHODS FOR IDENTIFYING MICROORGANISMS  With the development of molecular biology, the possi- bilities for identifying microorganisms have significant- ly expanded. Many microbiological laboratories still use  phenotypic and biochemical methods to identify microor- ganisms. However, the high specificity of molecular meth- ods helps to accurately and quickly identify various culti- vated and uncultivated species of microorganisms in any  samples without the need to grow them on nutrient media in laboratories. Notably, the significant development of molecular methods helped to launch a large-scale study of the microbiome and its impact on human health in 2007, called the Human Microbiome project [30].  The polymerase chain reaction (PCR) method has be- come the gold standard in microbiological laboratories,  which is used to identify microorganisms. The standard PCR method is based on the identification of bacterial DNA by increasing certain fragments of nucleic acids, that is, their amplification, followed by sequencing and comparing  them with databases [31]. Due to the wide variety of mi- croorganisms in biological samples, their identification by  cultural methods becomes difficult due to the inability to  isolate and identify uncultivated forms. PCR-based meth- ods not only provide rapid identification, but also help  identify microorganisms that are difficult to isolate in the laboratory [32]. However, despite its effectiveness, the PCR method has a number of disadvantages. The specificity of the search is that information can be obtained only about those microorganisms for which specific primers are used. PCR does not provide information about the viability of microorganisms, as it uses DNA or ribonucleic acid (RNA), which may be important in some cases. The lack of the possibility of preserving and further using microorganisms limits its use in some studies. In addition, it is worth noting that the method does not provide information about the sensitivity of microorganisms to antibiotics, which is an important aspect in clinical studies. DNA microarray. The use of DNA microarrays allows simultaneously identifying and sequencing hundreds or thousands of genes in a single study. A DNA microarray is a DNA probe attached to a microplate. The operation of a  DNA microarray is based on the phenomenon of hybridisa- tion, during which labelled samples are analysed with flu- orescent probes [33]. Unlike other molecular genetic meth- ods, DNA microarrays have a larger coverage area and faster  results. M.A. Campanero-Rhodes et al. [34] demonstrated the successful use of microarrays to detect type-specific immunoglobulin G (IgG) antibodies against Streptococcus pneumoniae capsule polysaccharide in serum (CPS). The  results showed the potential of microarrays for simultane- ous analysis of the interaction of multiple CPS using small  volumes of serum, which can be useful for studying lim- ited volumes of serum samples. In addition, according to  Hu. Arengaowa at al. [35], the DNA microarray method has been successfully used in the food industry to detect food  pathogens. Despite the successful use of the DNA microar- ray method, it has its drawbacks, which are expensive and  the need for high-quality DNA samples. Contamination or poor sample quality can lead to erroneous results, which complicates the research process. 16S rRNA sequencing is one of the most sensitive methods for detecting microorganisms, which is widely used in clinical settings. It is based on the detection of the  16S rRNA gene, which is specific for each type of microor- ganism [36]. This method provides accurate identification  of genera and species of microorganisms that do not meet any recognised biochemical profiles. A. Szymczak et al. [37] compared histological method, PCR, and 16S rRNA gene sequencing for identification of Helicobacter pylori. Using gastric antral biopsy, the researchers showed that 16S rRNA gene sequencing is the most sensitive method for detecting H. pylori. However, there are certain problems that can be encountered when using this method. One of them is that there are variations in the 16S rRNA gene among strains of the same species [38], which may affect the estimation of the abundance of these microorganisms.  Next-generation sequencing (NGS). Continuous im- provement of DNA sequencing technology has significantly  improved the capabilities of molecular genetic diagnostic methods. The main idea of using NGS is parallel sequencing of many fragments of DNA or RNA, which allows obtaining  significant amounts of genetic information in a short peri- od of time [39]. W. Gu et al. [40] developed a next-genera- tion metagenomic sequencing test (mNGS) using cell-free  Microbiological diagnostics...  38  Bulletin of Medical and Biological Research. 2023. Vol.5, No. 4 DNA from body fluids to identify pathogens. The results showed that rapid mNGS testing is an effective tool for diagnosing unknown infections. However, NGS research requires expensive equipment, timely and sustainable maintenance, and training of technical specialists, which can be a problem even for middle-income countries [41]. Whole-genome sequencing (WGS) is the most accurate method for identifying microorganisms and is an important  tool in microbiology [42]. This method expands the possi- bilities for analysing genes associated with antibiotic resist- ance [43], identifying their pathogenicity [44], and other key  aspects, making an important contribution to understand- ing microbial ecology. Despite these advantages, the meth- od has its drawbacks. It is considered the most expensive  and time-consuming; it does not provide information about the viability of microorganisms, since it is based on DNA/ RNA analysis. In addition, it provides only a correlation of  microorganisms, and identification problems are associat- ed with the lack of complete databases of genetic material.  Despite determining the microbial composition of an individual, it is important to understand the functions that bacteria perform in this community, their relationships, and their impact on the host [45]. The rapid development of molecular biology and information technology methods has contributed to the use of various omics technologies, such  as genomics, transcriptomics, proteomics, and metabolo- mics [46]. Omics technologies open up new opportunities  for cell research at more complex levels of life organisation.  Genomics is a set of methods that are based on the in- vestigation of the structure and functions of the genome of  the studied objects [47], whereas transcriptomics methods focus on studying the transcriptome of an organism, that is,  the sum of all its RNA transcripts [48]. Genomics and tran- scriptomics are two related but different areas of research  that help uncover and understand genetic information and  gene activity in the body. Proteomics examines the struc- ture and function of all proteins in the body [49]. It helps  to uncover the role of proteins in signalling interaction, vi- tal activity, and detection of diseases such as Alzheimer’s  disease [50]. Metabolomics represents the latest branch of omics technology and is extremely promising for medicine. The main task of metabolomics is to identify and quantify a  wide range of metabolites [51]. Detection of specific meta- bolic changes can serve as an indicator of the development  of various diseases, in particular, in diabetes mellitus [52] or in chronic obstructive pulmonary disease [53].  Investigation of the microbial composition of an or- ganism is a key to understanding the relationship between  microorganisms and their impact on the host. The use of  omics technologies provides new opportunities for stud- ying life systems at different levels of their organisation.  The integration of the data obtained with the help of these technologies helps to solve problems related to various pathological conditions of humans and the environment. e CONCLUSIONS  The study analysed and systematised the available meth- ods for identifying microorganisms in clinical diagnostics.  Analysing an overview of up-to-date data, it can be con- cluded that in everyday clinical practice, the microbio- logical method of isolating microbial cultures remains the  main one. This method not only determines the cause of  infection, but also helps to determine sensitivity to anti- bacterial drugs, contributing to the effective treatment of  various infections. However, cultural methods for studying  fastidious microorganisms are quite long and complex. Im- munological methods based on the interaction of antibod- ies with foreign antigens are widely used in microbiology  to identify individual species and serotypes of microorgan- isms. Despite their speed and simplicity of research, it is  important to consider some of the disadvantages of this  method. Immunological tests may be vulnerable to chang- es in the spectrum of antigens or antibodies, which can  lead to false results. In addition, the presence of chronic or immunodeficiency conditions can affect the accuracy and reliability of this method. Compared to traditional methods of microbiological diagnostics, molecular biology methods are characterised by higher sensitivity and specificity. The high sensitivity of these methods allows detecting microorganisms even with  a minimal number of them. In particular, the greater speci- ficity of molecular methods helps to more accurately iden- tify a specific type or strain of microorganism. However,  molecular genetic methods for identifying microorganisms also have drawbacks. High cost, expensive equipment, and  the need for specially trained personnel limit their availa- bility for many laboratories. In addition, molecular genet- ic methods do not provide information about whether the  microorganism is alive in the test sample.  Omics technologies (genomics, transcriptomics, pro- teomics, metabolomics) are a group of high-performance  methods that study large amounts of biological informa- tion at various levels of molecular structure and function,  which provides a deep understanding of complex biological  systems. The disadvantage of omics technologies and mo- lecular genetic methods is their high cost and the need for  complex equipment and interpreting large amounts of data requires specialised skills and bioinformatic knowledge. The results obtained contribute to the choice of a fast  and accurate method for identifying, classifying, and sys- tematising microorganisms in accordance with specific  needs. In particular, the ability to quickly identify microor- ganisms in various infectious conditions can significantly  speed up the choice of the optimal antibiotic therapy reg- imen for doctors and provide more effective treatment for  patients. Further research may include the development of new methods that combine the benefits of microbiological, immunological, molecular genetic, and omics technologies  to improve the accuracy, speed, and accessibility of infec- tious disease diagnostics.  e ACKNOWLEDGEMENTS None. e CONFLICT OF INTEREST The authors declare no conflict of interest.


REFERENCES

[1] Gao Y, Shang Q, Li W, Guo W, Stojadinovic A, Mannion C, et al. Antibiotics for cancer treatment: A double-edged sword. J Cancer. 2020;11(17):5135–49. DOI: 10.7150/jca.47470  M. Ivashko et al.  39  Bulletin of Medical and Biological Research. 2023. Vol.5, No. 4 [2] Meleshko T, Pallah O, Boyko N. Chapter 5 – Individual microbiota correction and human health: Programming and reprogramming of systemic and local immune response. In: Microbiome, Immunity, Digestive Health and Nutrition: Epidemiology, Pathophysiology, Prevention and Treatment. Academic Press; 2022. p. 53–67. DOI: 10.1016/B978-0- 12-822238-6.00015-7 [3] Farooqui T. Chapter 1 – Gut microbiota: Implications on human health and diseases. In: Gut Microbiota in Neurologic and Visceral Diseases. Academic Press; 2021. p. 1–27. DOI: 10.1016/B978-0-12-821039-0.00009-5 [4] Abushaheen MA, Muzaheed M, Fatani AJ, Alosaimi M, Mansy W, George M, et al. Antimicrobial resistance, mechanisms and its clinical significance. Dis Mon. 2020;66(6):e100971. DOI: 10.1016/j.disamonth.2020.100971 [5] Maugeri A, Barchitta M, Puglisi F, Agodi A. Socio-economic, governance and health indicators shaping antimicrobial resistance: An ecological analysis of 30 European countries. Glob Health. 2023;19:e12. DOI: 10.1186/s12992-023- 00913-0 [6] Salmanov A, Rudenko A. Antimicrobial resistance of leading uropathogens in Kyiv (Ukraine). Int J Antibiot Probiotics. 2017;1(2):48–60. DOI: 10.31405/ijap.1-2.17.03 [7] Hanišáková N, Vítězová M, Rittmann SK-MR. The historical development of cultivation techniques for methanogens and other strict anaerobes and their application in modern microbiology. Microorganisms. 2022;10(2):e412. DOI: 10.3390/microorganisms10020412 [8] Bororova O, Dziublyk Y, Iachnyk V. Modern methods of etiological diagnosing of acute community-acquired lower respiratory tract infections. Ukr Pulmonol J. 2021;29(3):58–65. DOI: 10.31215/2306-4927-2021-29-3-58-65 [9] Shevchenko M, Tyshkivska N, Andriychuk A, Martynenko O, Tsarenko T. Intralaboratory testing of the PCR protocol for molecular genetic identification of bacteria of the genus Staphylococcus spp. Sci J Vet Med. 2022;(1):81–91. DOI: 10.33245/2310-4902-2022-173-1-81-91 [10] Symonenko R. Modern methods of diagnosing periodontal tissue diseases in the concept of a systemic approach to treatment. (Literature review. Part 1). Actual Dent. 2023;(6):14–21. DOI: 10.33295/1992-576X-2023-6-14 [11] Trubka I, Korniienko L, Gosteva Z, Ermakova L, Zinkovych I, Stulikova V. Results of molecular genetic diagnosis of periodontal pathogens in young patients with rapidly aggressive periodontitis. Stomatol Bull. 2022;119(2):33–38. DOI: 10.35220/2078-8916-2022-44-2.6 [12] Motronenko V, Vlasiuk T. Laboratory diagnostics of resistant forms of tuberculosis – problems and prospects. Biomed Eng Technol. 2023;12(4):23–32. DOI: 10.20535/2617-8974.2023.12.291958 [13] Sethi S, Singh S, Banga SS, Jain N, Gupta S, Sharma N, et al. Revisiting blood agar for the isolation of Neisseria gonorrhoeae. Indian J Sex Transm Dis AIDS. 2020;41(2):221–22. DOI: 10.4103/ijstd.IJSTD_51_18 [14] Sanmak E, Aksaray S. Comparison of chromogenic culture media, rapid immunochromatographic test and temocillin resistance for the detection of OXA-48 carbapenemase-positive Klebsiella pneumonia strains. J Clin Exp Invest. 2021;12(4):em00778. DOI: 10.29333/jcei/11267 [15] Ferone M, Gowen A, Fanning S, Scannell AGM. Microbial detection and identification methods: Bench top assays to omics approaches. Compr Rev Food Sci Food Saf. 2020;19(6):3106–29. DOI: 10.1111/1541-4337.12618 [16] Thakur S, Joshi J, Kaur S. Leishmaniasis diagnosis: An update on the use of parasitological, immunological and molecular methods. J Parasit Dis. 2020;44:253–72. DOI: 10.1007/S12639-020-01212-W [17] Tapia Rico G, Chan MM, Loo KF. The safety and efficacy of immune checkpoint inhibitors in patients with advanced cancers and pre-existing chronic viral infections (Hepatitis B/C, HIV): A review of the available evidence. Cancer Treat Rev. 2020;86:e102011. DOI: 10.1016/J.CTRV.2020.102011 [18] Mahmud I, Garrett TJ. Mass spectrometry techniques in emerging pathogens studies: COVID-19 perspectives. J Am Soc Mass Spectrom. 2020;31(10):2013–24. DOI: 10.1021/JASMS.0C00238 [19] Sun Y, Stransky S, Aguilan J, Koul S, Garforth SJ, Brenowitz M, Sidoli S. High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry. Anal Chim Acta. 2021;1180:e338880. DOI: 10.1016/J.ACA.2021.338880 [20] Fialkov AB, Lehotay SJ, Amirav A. Less than one minute low-pressure gas chromatography – mass spectrometry. J. Chromatogr. A. 2020;1612:e460691. DOI: 10.1016/j.chroma.2019.460691  [21] Putri SP, Ikram MMM, Sato A, Dahlan HA, Rahmawati D, Ohto Y, Fukusaki E. Application of gas chromatography- mass spectrometry-based metabolomics in food science and technology. J Biosci Bioeng. 2022;133(5):425–35.  DOI: 10.1016/J.JBIOSC.2022.01.011 [22] Mohammad K, Jiang H, Titorenko VI. Quantitative metabolomics of saccharomyces cerevisiae using liquid chromatography coupled with tandem mass spectrometry. J Vis Exp. 2021;167. DOI: 10.3791/62061 [23] Jang KS, Kim YH. Rapid and robust MALDI-TOF MS techniques for microbial identification: A brief overview of their diverse applications. J Microbiol. 2018;56(4):209–16. DOI: 10.1007/s12275-018-7457-0 [24] Welker M, Van Belkum A, Girard V, Charrier J-P, Pincus D. An update on the routine application of MALDI-TOF MS in clinical microbiology. Expert Rev Proteomics. 2019;16(8):695–10. DOI: 10.1080/14789450.2019.1645603 [25] Do T, Guran R, Adam V, Zitka O. Use of MALDI-TOF mass spectrometry for virus identification: A review. Analyst. 2022;147(14):3131–54. DOI: 10.1039/D2AN00431C [26] Topić Popović N, Kazazić SP, Bojanić K, Strunjak-Perović I, Čož-Rakovac R. Sample preparation and culture condition effects on MALDI-TOF MS identification of bacteria: A review. Mass Spectrom Rev. 2023;42(5):1589–3. DOI: 10.1002/ mas.21739 [27] Doellinger J, Schneider A, Stark TD, Ehling-Schulz M, Lasch P. Evaluation of MALDI-ToF mass spectrometry for rapid detection of cereulide from Bacillus cereus cultures. Front Microbiol. 2020;11:e511674. DOI: 10.3389/ fmicb.2020.511674  Microbiological diagnostics...  40  Bulletin of Medical and Biological Research. 2023. Vol.5, No. 4 [28] Idelevich EA, Sparbier K, Kostrzewa M, Becker K. Rapid detection of antibiotic resistance by MALDI-TOF mass spectrometry using a novel direct-on-target microdroplet growth assay. Clin Microbiol Infect. 2018;24(7):738–43. DOI: 10.1016/j.cmi.2017.10.016 [29] Li D, Yi J, Han G, Qiao L. MALDI-TOF mass spectrometry in clinical analysis and research. ACS Meas. Sci. Au. 2022;2(5):385–4. DOI: 10.1021/acsmeasuresciau.2c00019 [30] Muller E, Algavi YM, Borenstein E. The gut microbiome-metabolome dataset collection: A curated resource for integrative meta-analysis. npj Biofilms Microbiomes. 2022;8:e79. DOI: 10.1038/s41522-022-00345-5 [31] Zhu H, Zhang H, Xu Y, Laššáková S, Korabečná M, Neužil P. PCR past, present and future. Biotechniques. 2020;69(4):317– 25. DOI: 10.2144/btn-2020-0057 [32] Salipante SJ, Jerome KR. Digital PCR – an emerging technology with broad applications in microbiology. Clin Chem. 2020;66(1):117–23. DOI: 10.1373/clinchem.2019.304048 [33] Babichev S, Khamula O, Perova I, Durnyak B. An analysis of gene regulatory network topology using results of DNA microchip experiments. In: Shakhovska N, Medykovskyy MO, editors. Advances in Intelligent Systems and Computing V. CSIT 2020; 2020 Sep 23-26; Zbarazh. Cham: Springer; 2021. p. 130–144. DOI: 10.1007/978-3-030-63270-0_9 [34] Campanero-Rhodes MA, Lacoma A, Prat C, García E, Solís D. Development and evaluation of a microarray platform for detection of serum antibodies against Streptococcus pneumoniae capsular polysaccharides. Anal Chem. 2020;92(11):7437–43. DOI: 10.1021/acs.analchem.0c01009 [35] Arengaowa, Hu W, Feng K, Jiang A, Xiu Z, Lao Y, et al. An in situ-synthesized gene chip for the detection of food-borne pathogens on fresh-cut cantaloupe and lettuce. Front Microbiol. 2019;10:e3089. DOI: 10.3389/ fmicb.2019.03089 [36] Church DL, Cerutti L, Gürtler A, Griener T, Zelazny A, Emler S. Performance and application of 16S rRNA gene cycle sequencing for routine identification of bacteria in the clinical microbiology laboratory. Clin Microbiol Rev. 2020;33(4). DOI: 10.1128/cmr.00053-19 [37] Szymczak A, Ferenc S, Majewska J, Miernikiewicz P, Gnus J, Witkiewicz W, Dąbrowska K. Application of 16S rRNA gene sequencing in Helicobacter pylori detection. PeerJ. 2020;8:e9099. DOI: 10.7717/peerj.9099 [38] Regueira-Iglesias A, Vázquez-González L, Balsa-Castro C, Blanco-Pintos T, Vila-Blanco N, Carreira MJ, Tomás I. Impact of 16S rRNA gene redundancy and primer pair selection on the quantification and classification of oral microbiota in next-generation sequencing [Internet]. Research Square [Preprint]. 2021 [cited 2024 Feb 23]. Available from: https://www.researchsquare.com/article/rs-662236/v1 [39] Hu T, Chitnis N, Monos D, Dinh A. Next-generation sequencing technologies: An overview. Hum Immunol. 2021;82(11):801–11. DOI: 10.1016/j.humimm.2021.02.012 [40] Gu W, Deng X, Lee M, Sucu YD, Arevalo S, Stryke D, et al. Rapid pathogen detection by metagenomic next-generation sequencing of infected body fluids. Nat Med. 2021;27:115–24. DOI: 10.1038/s41591-020-1105-z [41] Wilson ML, Fleming KA, Kuti MA, Looi LM, Lago N, Ru K. Access to pathology and laboratory medicine services: A crucial gap. Lancet. 2018;391(10133):1927–38. DOI: 10.1016/S0140-6736(18)30458-6 [42] Balloux F, Brønstad Brynildsrud O, van Dorp L, Shaw LP, Chen H, Harris KA, Wang H, Eldholm V. From theory to practice: Translating whole-genome sequencing (WGS) into the clinic. Trends Microbiol. 2018;26(12):1035–48. DOI: 10.1016/j.tim.2018.08.004 [43] Quainoo S, Coolen JPM, van Hijum SAFT, Huynen MA, Melchers WJG, et al. Whole-genome sequencing of bacterial pathogens: The future of nosocomial outbreak analysis. Clin Microbiol Rev. 2017;30(4):1015–63. DOI: 10.1128/ CMR.00016-17 [44] Satam H, Joshi K, Mangrolia U, Waghoo S, Zaidi G, Rawool S, et al. Next-generation sequencing technology: Current trends and advancements. Biology. 2023;12(7):e997. DOI: 10.3390/biology12070997 [45] Ogunrinola GA, Oyewale JO, Oshamika OO, Olasehinde GI. The human microbiome and its impacts on health. Int J Microbiol. 2020;2020:e8045646. DOI: 10.1155/2020/8045646  [46] Kumari A, Pooja, Sharma S, Acharya A. Introduction to molecular imaging, diagnostics, and therapy. In: Nanomaterial- Based Biomedical Applications in Molecular Imaging, Diagnostics and Therapy. Singapore: Springer; 2020. p. 11–26.  DOI: 10.1007/978-981-15-4280-0_2 [47] Koumakis L. Deep learning models in genomics: Are we there yet? Comput Struct Biotechnol J. 2020;18:1466–73. DOI: 10.1016/j.csbj.2020.06.017 [48] Prieto-Vila M, Yamamoto Y, Takahashi R, Ochiya T. Single-cell transcriptomics. In: Handbook of Single-Cell Technologies. Singapore: Springer; 2021. p. 585–6. DOI: 10.1007/978-981-10-8953-4_12 [49] McArdle AJ, Menikou S. What is proteomics? Arch Dis Child Educ Pract Ed. 2021;106(3):178–81. DOI: 10.1136/ archdischild-2019-317434 [50] Wingo AP, Liu Y, Gerasimov ES, Gockley J, Logsdon BA, Duong DM, et al. Integrating human brain proteomes with genome-wide association data implicates new proteins in Alzheimer’s disease pathogenesis. Nat Genet. 2021;53(2):143–46. DOI: 10.1038/s41588-020-00773-z [51] David A, Rostkowski P. Chapter 2 – Analytical techniques in metabolomics. In: Environmental Metabolomics. Elsevier; 2020. p. 35–64. DOI: 10.1016/b978-0-12-818196-6.00002-9 [52] Qiu S, Wang W, Zhang A, Wang X. Mass spectrometry-based metabolomics toward biological function analysis. In: Mass Spectrometry-Based Metabolomics in Clinical and Herbal Medicines. Wiley; 2021. p. 157–70. DOI: 10.1002/9783527835751.ch12 [53] Ran N, Pang Z, Gu Y, Pan H, Zuo X, Guan X, et al. An updated overview of metabolomic profile changes in chronic obstructive pulmonary disease. Metabolites. 2019; 9(6):e111. DOI: 10.3390/metabo9060111  M. Ivashko et al.  41  Bulletin of Medical and Biological Research. 2023. Vol.5, No. 4 


Мікробіологічна діагностика: від традиційних до молекулярно-генетичних методів: огляд літератури Максим Васильович Івашко Аспірант, асистент Державний вищий навчальний заклад «Ужгородський національний університет» 88000, пл. Народна, 3, м. Ужгород, Україна https://orcid.org/0009-0007-4964-2417 Світлана Андріївна Бурмей Аспірант, асистент Державний вищий навчальний заклад «Ужгородський національний університет» 88000, пл. Народна, 3, м. Ужгород, Україна https://orcid.org/0000-0002-8157-4262 Леся Сергіївна Юсько Кандидат біологічних наук, доцент Державний вищий навчальний заклад «Ужгородський національний університет» 88000, пл. Народна, 3, м. Ужгород, Україна https://orcid.org/0000-0002-7072-0703 Тетяна Василівна Чайковська Кандидат медичних наук, доцент Державний вищий навчальний заклад «Ужгородський національний університет» 88000, пл. Народна, 3, м. Ужгород, Україна https://orcid.org/0009-0000-2008-4270 Надія Володимирівна Бойко Доктор біологічних наук, професор Державний вищий навчальний заклад «Ужгородський національний університет» 88000, пл. Народна, 3, м. Ужгород, Україна https://orcid.org/0000-0002-2467-7513 Анотація. Розробка нових та оптимізація відомих культуральних та молекулярно-генетичних методів для точної видової ідентифікації мікроорганізмів є актуальним і практично необхідним завданням, якому приділяється велика увага дослідників. Метою даної роботи було проведення аналізу, систематизація теоретичних наукових даних щодо методів ідентифікації мікроорганізмів та оцінка їхніх основних переваг і недоліків. Для цього було здійснено систематичний огляд рандомізованих 53 наукових робіт, опублікованих в період з 2018 по 2023 роки. Пошук публікацій із використанням ключових термінів «мікробіом», «мікробіологічна діагностика», «ідентифікація мікроорганізмів», «секвенування» та «омікс-технології» у назві чи тексті науково-дослідницької роботи здійснювали в системах Web of Science, Scopus, PubMed та Google Scholar. У статті надано узагальнену інформацію щодо традиційних та сучасних методів ідентифікації мікроорганізмів. Встановлено, що до переваг традиційних методів діагностики належить можливість збереження отриманих мікроорганізмів для подальших досліджень, зокрема для визначення їх чутливості до антибіотиків. Сучасні молекулярно-генетичні методи відкривають нові можливості для точної ідентифікації мікроорганізмів, зокрема тих, які складно культивувати за допомогою традиційних культуральних методів. Використання цих технік дозволяє отримати детальні дані про генетичну структуру та різноманіття мікроорганізмів, що є важливим у багатьох галузях, включаючи мікробіологію, медицину та екологію. Проте, молекулярні методи більш чутливі до забруднень чи похибок у процесі збирання та обробки зразків. Варто також відмітити omics-технології (геноміка, транскриптоміка, протеоміка та метаболоміка), які відкривають нові можливості для дослідження клітин на більш складних рівнях організації життя. В повсякденній клінічній практиці основним залишається мікробіологічний метод виділення мікробних культур, оскільки тільки він дозволяє визначити не лише причину інфекції, але й чутливість до антибіотиків. У той же час культуральні методи дослідження вибагливих мікроорганізмів досить обмежені. Таким чином, отримані результати сприяють розробці швидкої та точної методики ідентифікації, класифікації та систематизації мікроорганізмів, що полегшує процеси діагностики та розробки стратегій контролю і лікування інфекційних захворювань у лабораторіях та клінічних установах Ключові слова: мікробіологія; діагностика; поживне середовище; ідентифікація; секвенування; омікс-технології



Коментарі

Оцінка: 0 з 5 зірок.
Ще немає оцінок
Додайте оцінку
bottom of page