IRN AP27510713 Project topic «Assessment of the prevalence of antibiotic-resistant conditionally pathogenic bacteria in surface and groundwater ecosystems»

Implementation period 2025-2027

Relevance

Bacterial resistance to antibiotics has been a growing concern in various areas of human activity worldwide in recent years. Incorrect and excessive use of antimicrobial drugs has contributed to the development of specific resistance in bacteria, which is formed as a result of the presence of special genes responsible for antibiotic resistance in the bacterial genome. Some representatives of the bacterial domain exhibit multiple resistance to antibiotics, which complicates the process and measures to combat these representatives of pathogenic microorganisms. At the same time, microorganisms, and in particular bacteria, are ubiquitous in nature, which undoubtedly increases the level of potential risk and threat to human and animal health. In addition, the main favorable environment for the vital activity and reproduction of bacteria is inherently water and the aquatic environment. The rate of spread of microorganisms in water is much higher than in any other natural substrate, and the constant water cycle contributes to the rapid and widespread spread of the bacterial community in natural conditions. In connection with the above, water samples from natural sources will be used to isolate antibiotic-resistant conditionally pathogenic bacteria during the implementation of the presented research project. The idea of the project is to assess the prevalence of multi-resistant conditionally pathogenic bacteria in natural ecosystems of surface and groundwater in the West Kazakhstan Region (WKR). The project is based on comprehensive studies to identify bacteria, determine their biochemical characteristics, assess the response to the effects of antimicrobial drugs, considering the identification of responsible genes, analyze virulence factors, and identify the risk of negative impact of bacteria with multiple antibiotic resistance on the environment. The obtained results of the distribution area, species, biochemical characteristics, analysis of resistance to antimicrobial drugs, as well as the virulence of isolated bacterial strains will allow us to assess the level of anthropogenic impact on the microbiota of natural water sources, in order to develop recommendations for regulating the use of antimicrobial drugs. As a result of the research, a map of the distribution of conditionally pathogenic, antibiotic-resistant strains of bacteria in natural water sources of the West Kazakhstan Region will be created, with a complete biological description of the species, including biochemical and molecular genetic identification, distribution area, antibiotic resistance and virulence factors.

Project goal

To investigate the prevalence and diversity of antibiotic-resistant conditionally pathogenic bacteria in surface and groundwater ecosystems and to determine the potential environmental risk of isolated bacterial strains by assessing their antibiotic resistance, biochemical characteristics and virulence factors.

Project objectives

Task 1. Creation of a collection of cultures of conditionally pathogenic bacteria included in the class Gammaproteobacteria isolated from the selected water samples of the ecosystem of surface and groundwater of the West Kazakhstan Region. This task has three main objectives: (i) Collection of water samples from surface and groundwater ecosystems of the West Kazakhstan region, followed by creation of a map of the collected samples. (ii) Isolation of "pure" bacterial cultures on liquid and solid nutrient media. (iii) Determination of the morphology and cultural properties of isolated bacterial colonies. Completion of task 1 will allow us to create our own collection of conditionally pathogenic bacteria cultures for subsequent research.

Task 2. Identification of isolated colonies of conditionally pathogenic bacteria belonging to the class Gammaproteobacteria. This task has two main goals: (i) Determination of the genus of isolated bacterial colonies as a result of evaluation of biochemical characteristics. (ii) Molecular genetic identification of bacterial isolates based on the PCR method using genus-specific primer pairs, as well as sequencing of the 16S rRNA gene. When completing task 2, the genus and species of isolated bacterial strains will be identified.

Task 3. To assess the prevalence of antibiotic-resistant bacteria belonging to the class Gammaproteobacteria in natural water sources located within the territory of the West Kazakhstan region. This task has two main goals: (i) To determine the resistance of isolated bacterial strains to β-lactam antibiotics, as well as antibiotics of the tetracycline, aminoglycoside and amphenicol groups. (ii) Analysis of the presence of genes responsible for antibiotic resistance in the genome of bacterial strains. Solving problem 3 will allow us to determine the breadth of distribution of bacteria exhibiting multiple antibiotic resistance in the natural environment.

Task 4. Determination of virulence factors in isolated bacterial strains. Completion of task 4 will allow us to identify the main pathogenicity genes in isolated antibiotic-resistant strains of surface and ground waters, as well as to determine the potential risk to humans and animals.

Expected results

The research will result in the following: - creation of collections of cultures of conditionally pathogenic bacteria belonging to the class Gammaproteobacteria isolated from the selected water samples of the surface and groundwater ecosystem of the West Kazakhstan region; - collection of water samples from the surface and groundwater ecosystems of WKR. A map of the selected samples will be created. 60 samples will be collected from at least 10 water bodies of WKR (1 sample from the surface, 1 sample from the depth from 3 points of one water body) and 30 samples from wells of 10 settlements of WKR, indicating the geographic location of the sampling points; - isolation of "pure" bacterial cultures on liquid and solid nutrient media belonging to the class Gammaproteobacteria. Optimal growing conditions will be determined; - morphological properties of isolated bacterial colonies will be determined. The following properties will be determined: shape, size, surface, profile, transparency, color, structure and consistency; - the isolated colonies of conditionally pathogenic bacteria belonging to the class Gammaproteobacteria will be identified; - the genus of the isolated bacterial colonies will be determined based on the assessment of their biochemical characteristics; - molecular genetic identification of bacterial isolates will be carried out based on the PCR method using genus-specific primer pairs, as well as sequencing of the 16S rRNA gene; - an assessment will be made of the prevalence of antibiotic-resistant bacteria belonging to the class Gammaproteobacteria in natural water sources located within the territory of the West Kazakhstan region; - the resistance of isolated bacterial strains to β-lactam antibiotics, as well as antibiotics of the tetracycline, aminoglycoside and amphenicol groups will be determined; - an analysis will be conducted of the presence of genes (acrD, aadA, aadB, blaKPC, qnrA, sul1, tet(A) and mecA) responsible for antibiotic resistance in the DNA of bacterial strains; - virulence factors in isolated bacterial strains will be determined. Whole genome sequencing will be carried out. The following will be published:

- at least 2 (two) articles and (or) reviews in peer-reviewed scientific publications in the scientific area of the project, indexed in the Science Citation Index Expanded and included in the 1st (first), 2nd (second) or 3rd (third) quartile by impact factor in the Web of Science database and (or) having a CiteScore percentile in the Scopus database of at least 50 (fifty);

- at least 2 (two) articles or reviews in a peer-reviewed foreign or domestic publication recommended from lists 1 and 2 of the SHEQAC;

- either at least 1 (one) article or review in a peer-reviewed scientific publication indexed in the Science Citation Index Expanded and included in the 1st (first) quartile by impact factor in the Web of Science database and (or) having a CiteScore percentile in the Scopus database of at least 80 (eighty).

- at least 1 (one) article or review in a peer-reviewed foreign or domestic publication recommended from lists 1 and 2 of the SHEQAC.

Results achieved

A collection of cultures of conditionally pathogenic bacteria belonging to the class Gammaproteobacteria was established from isolates obtained from water samples of surface and groundwater ecosystems of the West Kazakhstan Region.

Sampling of water from surface and groundwater ecosystems of the West Kazakhstan Region (WKR) was carried out. A map of the sampled sites was created. In total, 216 water samples were collected from 12 water bodies of the WKR (1 surface sample and 1 bottom sample from 9 points within each water body), as well as 30 samples from wells in 10 settlements of the WKR, with geographic coordinates of all sampling points indicated. As a result of the analysis, the following lakes of the WKR were studied. From the steppe zone: Brusyanoye (51°21.426′ N, 051°50.913′ E), Prorva (51°14.573′ N, 051°31.220′ E), Zhaltyrkol (50°59.388′ N, 051°25.864′ E), Zhamankara (50°56.147′ N, 051°51.704′ E); from the semi-desert zone: Kolovernoye (50°36.013′ N, 051°06.764′ E), Glubinnoye (50°19.630′ N, 051°03.445′ E), Rybny Sakryl (49°37.871′ N, 049°21.447′ E), Saryshyganak (49°27.901′ N, 049°51.589′ E); from the desert zone: Edilsor (49°13.361′ N, 050°44.029′ E), Aydyn (48°57.133′ N, 049°35.183′ E), Karasu (48°49.407′ N, 051°52.274′ E), Tasonqar (49°21.311′ N, 050°22.046′ E). In addition, the following settlements were studied: Ozernoye (51°21.724′ N, 051°50.272′ E), Zhelayevo (51°15.122′ N, 051°30.007′ E), Kemer (51°01.585′ N, 051°24.870′ E), Bekey (50°57.435′ N, 051°46.805′ E), Kolovernoye (50°35.613′ N, 051°06.645′ E), Zhanabulak (50°18.953′ N, 051°03.915′ E), Zhalpaktal (49°35.003′ N, 049°21.265′ E), Kosoba (49°27.177′ N, 050°17.572′ E), Kyzylbas (49°25.874′ N, 050°15.380′ E), and Zhangala (49°13.202′ N, 050°17.460′ E). The studied water bodies differ significantly in their physicochemical characteristics, which is related to their geographic location, depth, type of bottom sediments, and degree of mineralization. Most of the water bodies have an alkaline environment (pH from 8.0 to 9.6), which is typical for steppe and brackish lakes. The level of dissolved oxygen varies widely—from extremely low values (2.5–4.1 mg/L in Lake Saryshyganak) to very high values (over 20 mg/L in Lake Tasonqar). This may indicate both oxygen depletion due to organic pollution and active photosynthesis under conditions of high temperature and solar radiation. Water temperature also differs noticeably among the water bodies: the lowest values are observed in Lakes Brusyanoye and Zhamankara (about 20–21 °C), while the highest are recorded in Lakes Prorva, Glubinnoye, Tasonqar, and Kolovernoye (up to 27 °C), which may be associated with their geographic location and shallow depth. Water mineralization shows substantial variation, ranging from very weakly mineralized waters (1.6–3.7 mg/L in Lakes Saryshyganak, Karasu, and Aydyn) to highly mineralized water bodies such as Tasonqar (up to 10 mg/L) and Zhaltyrkol (up to 528.7 mg/L). Electrical conductivity also varies from very low values (about 3–7.5 µS/cm in brackish lakes such as Rybny Sakryl, Saryshyganak, Edilsor, Karasu, and Aydyn) to moderate values (28.1–174.1 µS/cm in Lakes Tasonqar and Zhamankara) and high values (up to 948.7 µS/cm in lakes with high salt and organic content, such as Zhaltyrkol, Brusyanoye, Prorva, Kolovernoye, and Glubinnoye). Water transparency is low in most water bodies, indicating a high degree of turbidity or the presence of suspended solids. Lakes with black or gray silty bottom sediments generally exhibit lower transparency and higher indicators of organic pollution. Sandy bottoms (for example, in Lakes Prorva and Zhamankara) are associated with higher transparency and better aeration. Overall, the best oxygen saturation among all water bodies is observed in Tasonqar, Edilsor, and Kolovernoye, while the poorest conditions are recorded in Saryshyganak and Rybny Sakryl, which may indicate an unfavorable ecological status of the latter. Water from wells in different settlements shows considerable variability in physicochemical parameters. Water temperature in wells ranges from 14.3 to 17.2 °C, which is typical for groundwater and depends on depth and seasonal conditions. Dissolved oxygen concentration varies widely—from low values of about 1.4 mg/L in Kyzylbas to high values exceeding 10 mg/L in Zhelayevo and Zhanabulak—reflecting differences in aeration and oxygen penetration into aquifers. The percentage of oxygen saturation ranges broadly (from 13.4% to 110%), indicating both stagnant or polluted areas and fresher, better-aerated waters. Mineralization of well water differs among settlements: in some wells it is relatively high (around 460 mg/L in Ozernoye, Zhelayevo, and Zhalpaktal), indicating a significant content of dissolved salts, while in others it is much lower (1.4–4 mg/L in Kemer and Kolovernoye). Salinity ranges from very low (0.25‰ in Kyzylbas) to moderate (4.6‰ in Zhangala), also reflecting differences in groundwater composition. pH values are predominantly neutral or slightly alkaline (7.2–8.35), which is typical for groundwater and generally favorable for drinking purposes. Electrical conductivity ranges from low values (about 2.6 µS/cm in Kolovernoye) to very high values (over 1600 µS/cm in Kosoba), reflecting differences in ionic composition and mineralization. Well depth varies from shallow (3–5 m: Kosoba, Kyzylbas, Zhangala) and intermediate (7.5–9.7 m: Bekey, Zhalpaktal, Ozernoye) to deeper wells (over 20 m: Zhelayevo, Kemer, Kolovernoye, Zhanabulak), which influences water quality and physicochemical characteristics. Overall, the data indicate significant heterogeneity in groundwater quality among different settlements, which should be taken into account when using these waters for drinking and household needs.

“Pure” bacterial cultures belonging to the class Gammaproteobacteria were isolated on liquid and solid nutrient media, and their optimal growth conditions were determined. As a result of the study, 1,080 and 150 pure bacterial cultures were isolated from water samples taken from lakes and wells, respectively. The experiments were carried out using Luria–Bertani (LB) medium, both with and without agar. When cultivating bacteria in liquid LB medium, continuous stirring and aeration were applied. It was established that the best growth of the cultures was observed at a temperature of 30–37 °C and a neutral pH level (7.0–7.2). Maintaining aeration ensured active cell reproduction and stable metabolic activity, which contributed to an increase in biomass and an acceleration of the exponential growth phase.

The morphological properties of the isolated bacterial colonies were determined. The following characteristics were assessed: shape, size, surface, elevation (profile), transparency, color, margin type, structure, and consistency. As a result of the analysis of 1,080 bacterial colonies isolated from lake water, the following characteristics were identified. Regarding shape, 1,040 colonies (96.3%) were circular, while 40 (3.7%) were amoeboid. In terms of size, the distribution was as follows: 0.5 mm – 39 (3.61%), 1 mm – 198 (18.33%), 1.5 mm – 319 (29.54%), 2 mm – 154 (14.26%), 2.5 mm – 175 (16.2%), 3 mm – 72 (6.67%), 3.5 mm – 45 (4.17%), 4 mm – 22 (2.04%), 4.5 mm – 28 (2.59%), 5 mm – 13 (1.2%), 5.5 mm – 5 (0.46%), 6 mm – 3 (0.28%), 7 mm – 1 (0.09%), 7.5 mm – 1 (0.09%), 8 mm – 1 (0.09%), 10 mm – 2 (0.19%), and 12 mm – 2 (0.19%). With respect to surface characteristics, 967 colonies (89.5%) had a smooth surface, whereas 113 (10.5%) were rough. The colony profile was predominantly convex, observed in 1,075 colonies (99.5%), while only 5 colonies (0.5%) were flat. In terms of transparency, most colonies were opaque—1,041 (96.4%), with 25 (2.3%) being semi-transparent and 14 (1.3%) transparent. Regarding color, the colonies were mainly beige—583 (54%), light gray—353 (32.69%), yellow—90 (8.33%), and white—46 (4.25%). Less frequently observed colors included light brown—1 (0.09%), beige-gray—1 (0.09%), pink—5 (0.46%), and light pink—1 (0.09%). The colony margins were predominantly smooth—908 (84.1%), followed by wavy—171 (15.8%), and rarely serrated—1 (0.1%). All colonies (100%) had a homogeneous structure. In terms of consistency, 1,076 colonies (99.6%) were soft, while 4 (0.4%) were firm. For the 150 bacterial colonies isolated from well water, the following morphological characteristics were recorded. In terms of shape, 143 colonies (95.3%) were circular and 7 (4.7%) were amoeboid. The size distribution was as follows: 0.5 mm – 12 (8%), 1 mm – 36 (24%), 1.5 mm – 29 (19.33%), 2 mm – 24 (16%), 2.5 mm – 30 (20%), 3 mm – 6 (4%), 3.5 mm – 3 (2%), 4 mm – 1 (0.67%), 4.5 mm – 2 (1.33%), 5 mm – 4 (2.67%), 5.5 mm – 1 (0.67%), 6 mm – 1 (0.67%), and 6.5 mm – 1 (0.67%). Regarding the surface, 137 colonies (91.3%) were smooth and 13 (8.7%) were rough. The profile was predominantly convex—149 colonies (99.3%), with only 1 colony (0.7%) being flat. In terms of transparency, 144 colonies (96%) were opaque, while 6 (4%) were semi-transparent. The color of the colonies was mainly beige—77 (51.3%), light gray—40 (26.7%), yellow—24 (16%), white—8 (5.3%), and gray—1 (0.7%). The margins were mostly smooth—139 (92.7%), with wavy edges observed in 11 colonies (7.3%). All colonies (100%) exhibited a homogeneous structure. With respect to consistency, 146 colonies (97.3%) were soft, while 4 (2.7%) were firm.

Bakiyev Serik Samigullovich – Project Leader, PhD.

Scopus Author ID,

(https://www.scopus.com/authid/detail.uri?authorId=57214922444).

Researcher ID, (AAM-9369-2020)

ORCID https://orcid.org/0000-0001-5095-6869