Contamination of Kazakhstan cheeses originating from Escherichia coli and its resistance to antimicrobial drugs

Background and Aim: Escherichia coli, a commensal intestine bacterium of vertebrates, is widely distributed in the environment and indicates the microbiological quality of food products in relation to coliforms. In addition, virulent strains, particularly E. coli O157:H7, cause outbreaks of toxic infections caused by consuming dairy products. Because food safety studies regarding E. coli have not been conducted in Central Asia, this research aimed to study the characteristics of contamination, microbiological and genotypic properties, and resistance to antimicrobial agents of E. coli strains that contaminate various types of commercialized cheeses originating from Kazakhstan. Materials and Methods: In retail outlets, 207 samples of three types of cheese produced by 22 industrial and eight small enterprises in the central, eastern, southern, and northern regions of Kazakhstan were selected in 2020–2023. E. coli contamination was examined using standard microbiological, mass spectrometric, and molecular genetic methods. The discodiffuse European Committee on Antimicrobial Susceptibility Testing method was used to test the resistance of the identified E. coli isolates (65/207; 31.4%) to 20 antibacterial drugs. The Shiga toxin-producing E. coli (VT1 and VT2) and E. coli O157:H7 (eae) genes were investigated in all E. coli isolates using multiplex polymerase chain reaction. Results: An average of 31.4% samples of commercial Kazakhstani cheeses of various types were found to be contaminated with E. coli in almost all geographical regions of Kazakhstan, regardless of the productivity of the dairy enterprises. Soft cheeses produced by small farms (80% of samples) packaged at the retail site (100%) were the most contaminated with E. coli. The microbiological index (colony-forming unit/g) was unsatisfactory and unsuitable in 6.2% of such cheese samples. For the first time in Central Asia, the enteropathogenic strain E. coli O157:H7 was detected in 0.5% of cheese samples. E. coli isolates from cheese samples were resistant to 65% of antibacterial drugs and contained resistance genes to β-lactams, sulfonamides, and quinolones groups. At the same time, 25% of the E. coli isolates were multi-resistant to three or more antimicrobial agents. Conclusion: The high level of contamination caused by multi-antibiotic resistant E. coli strains, including pathogenic pathogens, poses a risk to public health and highlights the need for further research on the monitoring and control of coliform enteropathogens in food products.


Introduction
There is an increase in global consumer demand for cheeses, which are considered the most useful source of vital nutrients, such as vitamins, minerals, and proteins, which constitute the main part of healthy foods [1].Cheese production is one of the main sectors of the dairy industry and cheeses are usually made from raw or pasteurized milk [2,3].According to the Bureau of National Statistics of Kazakhstan, in 2022, 38 thousand tons of cheese were produced in the country, the production of which has increased by 50% over the past 5 years due to the expansion of market capacity after the growth trend of urbanization.In general, the consumption of cheese is 2.1 kg/ person/year, and it is considered the preferred product in the everyday and festive diet of Kazakhstani consumers (www: statgov.kz).
Although cheeses are considered to be microbiologically safe food products, outbreaks of toxic and infectious diseases associated with the contamination of these dairy products with pathogenic bacteria have been regularly recorded in different regions of Copyright: Kuzeubayeva, et al.Open Access.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
the world [4].In this respect, cheeses made from raw milk are particularly dangerous [5].This indicates the urgency of the problem of bacterial contamination, given that, as mentioned above, global and domestic demand for these substances continues to grow.It has been shown that pathogenic bacteria in cheeses can persist due to their survival during production.In addition, dangerous microorganisms may enter the cheese at retail outlets where cutting and packaging take place, and sanitary conditions are not sufficient to prevent re-contamination [6,7].
Escherichia coli, a Gram-negative, non-spore-forming bacterium of the Enterobacteriaceae family [8], is the most common microorganism transmitted through dairy products.In addition to Escherichia, the common genera of this family, which also contaminate cheeses and other products, are Citrobacter, Enterobacter, Klebsiella, etc. [9][10][11].It is well known that samples of dairy products contaminated by E. coli usually contain a significantly higher number of these bacterial genera and the overall organoleptic characteristics of the products are low.Thus, E. coli serves as an indicator for assessing the microbiological quality of dairy products, indicating the presence of other food enteropathogenic pathogens [10,12].
E. coli is an important representative of the normal intestinal microflora of humans and other mammals [13].However, this species has at least six pathotypes that cause a variety of intestinal and extraintestinal disorders.According to the virulence factor, bacteria of this species can be divided into several groups, including enterotoxigenic E. coli, attaching and effacing E. coli, enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli, and Shiga toxin-producing E. coli (STEC)/verotoxins-producing E. coli [14].Cheese producers are concerned about E. coli, which produces shiga/verotoxins encoded by th STX gene [15,16].Sporadic infections caused by the E. coli O157:H7 strain have become an important cause of endemic disease outbreaks in Europe [17].E. coli pathotypes are recognized as significant foodborne pathogens; therefore, rapid identification of E. coli pathotypes is important for food hygiene management and rapid epidemiological actions.
In addition to traditional methods of microbiological cultivation based on morphological and biochemical characteristics, molecular biological methods have become a priority in the study of the Enterobacteriaceae family for species identification, epidemiological typing, and determination of a phylogenetic relationship between pathogenic and nonpathogenic bacteria.In addition, due to its practical significance, bacteria of this family can transfer and transmit genes of resistance to antibacterial drugs to other microorganisms in the human and animal gut microbiome [18].At present, antibiotic resistance is recognized as a significant public health problem and poses a threat to human health [19,20].
The epidemiological characteristics and antibiotic resistance of microbial contamination of cheeses with E. coli species in Central Asia have not yet been investigated.Therefore, it is of scientific and practical interest to identify the food safety of domestic producers' cheeses in relation to pathogenic strains of E. coli and other Enterobacteriaceae family microorganisms.
Therefore, this study aimed to identify the epidemiological features, phenotypic and genotypic properties, and resistance to antimicrobial agents of E. coli strains that contaminate various cheeses produced in Kazakhstan.

Ethical approval
The study goal does not require ethical approval because the study was conducted on cheese samples.

Study period and location
From September 2020 to April 2023, cheese samples were collected at retail outlets and farmers' markets in the central, eastern, southern, and northern regions of Kazakhstan (Figure -1 and Table-1).
Microbiological and molecular genetic studies were conducted at the Kazakh-Chinese Biosafety Laboratory of the S. Seifullin Kazakh Agrotechnical Research University, the Laboratory of Molecular Diagnostics and Food Safety of Serrahpasha Istanbul University, the Research Institute of Applied Biotechnology of the A. Baitursynov Kostanay Regional University, and the National Center of Biotechnology of Kazakhstan.

Sampling
In the studied regions, 207 samples of three types of cheese (hard, semi-hard, and soft) from 15 brands, 22 industrial enterprises, and eight small farm producers were collected by random selection.At the same time, 78% and 22% of the samples were sold in factory individual vacuum packaging and cut and placed in plastic bags after purchase, respectively (Table -1).
Samples collected under aseptic conditions were delivered to the laboratory in an ice box and microbiological examination was carried out on the day of admission.The remaining volume of samples was stored at −20°C for further research.

Microbiological studies
Primary microbiological studies of cheese samples for the presence of E. coli were performed using Compact Dry EC commercial plates (R-Biopharm AG, Japan) according to the instructions.
Mass spectrometric analysis of isolated bacterial cultures from 93 samples was performed in a linear mode on matrix-assisted laser desorption ionization (MALDI) Biotyper 4.0, Bruker Daltonics (Germany) for species identification.For this purpose, cultures of microorganisms (single colonies) were applied to the cells of a 96-well MSP chip (MSP 96 target polished steel BC, microScoutTarget).Next, 1 mL of matrix solution (saturated solution of α-Cyano-4-hydroxycoric acid with 50% acetonitrile and 2.5% trifluoroacetic acid) was applied and dried at 20-25 0 C. The chip deposited with the samples was placed in a MALDI-time of flight microflex LT mass spectrometer (Bruker Daltonics).After positioning the chip in the ionization chamber and reaching the required values of the operating parameters of the device, calibration was performed using the applied calibration standard.Then, the spectra were collected in automatic mode.To obtain a single mass spectrum, 40 laser pulses (60 Hz frequency) were used.The mass/charge range analyzed was 2000-20,000 Da.Mass spectra were recorded, processed, and analyzed using flexControl software, MALDI Biotyper version 3.0, and MALDI Biotyper RTC (Bruker Daltonik).

Identification of the pathogenic E. coli strains
Isolates from 65 cheese samples were tested for the presence of STEC and E. coli O157:H7 strains by determining STX (VT1 and VT2) and eae genes in DNA using known primers (Table -2) in multiplex polymerase chain reaction (PCR) according to the methodology described in the Terrestrial Manual of the World Organization for Animal Health [23].
For DNA extraction from E. coli colonies, the QIAmp DNA Mini Kit (Qiagen, Germany) was used according to the manufacturer's instructions.DNA concentration was measured using a NanoDrop 1000 spectrophotometer at a wavelength of 260 nm, and a qualitative assessment of DNA was performed by electrophoresis.

Detection of antibiotic resistance
The resistance of the identified E. coli isolates to antibacterial drugs was tested using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) Disk Diffusion Method for Antimicrobial Suspicion Testing Version 9.0 (January 2021) using Muller-Hinton agar (HiMedia Laboratories, India).The isolates were diluted in normal 0.85% saline solution to a density of 0. Isolates of bacteria that are resistant to at least three classes of antibiotics have been classified as multidrug-resistant microorganisms [24].
The reaction mixture consisted of DreamTaq Green Master Mix (Thermo Fisher Scientific Inc., USA), water, 10 pmol1 primers, and the study DNA.As shown in Table-4, the amplification mode was selected for each pair of primer.A 100 bp marker, Tris-borate-EDTA buffer solution, and SYBR Safe DNA gel stain dye (Thermo Fisher Scientific Inc.) were used to set the reaction conditions.Amplification products were detected by electrophoresis in 1.5% agarose gel (Thermo Fisher Scientific) using a Quantum ultraviolet transilluminator (1100SUPER-BRIGHT; Reqlab, Germany).

Results
After sowing on Compact Dry EC plate, cheeses from different regions of Kazakhstan, produced both in vacuum packaging and without packaging, colonies of blue color were isolated from 93 samples (44.9%), demonstrating the morphological characteristics of E. coli.Bacterial colonies of similar colors formed due to the chromogenic substrate 5-bromo-4-chloro-3-indolyl-B-D-glucuronide.At the same time, artisan cheeses produced using artisanal methods had the highest levels of contamination.
Spectrometric analysis of the isolated microorganisms on MALDI revealed that they were identified as species of the following enterobacteria genera: Escherichia, Enterobacter, and Citrobacter.E. coli was detected more frequently (69.9%), indicating post-pasteurization contamination of dairy products from the production environment.
Subsequent bacteriological studies of 65 samples of the cheese under study in which E. coli was spectrometrically identified confirmed the presence and allowed the isolation of colonies of morphologically and biochemically identifiable E. coli bacteria.
According to the Public Health Laboratories (PHLs) hygienic standards for colony-forming unit (CFU), a large proportion of E. coli-contaminated cheese samples were satisfactory (18.4%) and acceptable (10.6%), whereas 1.9 and 0.5% of cheese samples were unsatisfactory and unacceptable, respectively.Simultaneously, the last two categories of microbiological assessment were mainly characterized by soft (6.2%) and semi-hard (0.9%) types of cheeses .
Analysis of the geographical distribution of cheese contamination with E. coli showed that samples from  the northern region had the highest percentage of detectability of these bacteria, reaching 46% of the total number of isolates, whereas the proportion of contaminated samples from the eastern and central regions was lower (29% and 25%, respectively).E. coli strains were not found in cheese samples from the southern region.In total, 31.4% of product samples were contaminated by E. coli, with soft and semi-hard cheese varieties being the most contaminated (48 and 28%, respectively), while only 15% of hard product samples were contaminated.This distribution pattern of contamination by cheese varieties was typical for all country regions.The level of E. coli contamination of commercialized cheeses depended on the size of the enterprises.
Therefore, approximately two-thirds of the products studied from small farms were contaminated with E. coli (65%), and approximately one-third of the products of large and medium-sized enterprises were found to contain E. coli (27.5 and 31%, respectively).Simultaneously, contamination was most often observed in all enterprises with soft cheeses, and 80% of small artisanal cheese producers were contaminated .
When examining the isolates for the presence of pathogenic strains, it was found that during electrophoresis, the amplification products of the E. coli DNA primer sites of one soft cheese sample from a small farm produced unique bands of approximately 618 bp corresponding to the eae gene, proving the presence of the E. coli O157:H7 strain in the bacterial culture.
A study of E. coli strains for resistance to 20 antibacterial drugs showed that the isolates were sensitive only to drugs of the aminoglycoside group (STR, KN, GNT), 50% of the β-lactam group drugs (AMP, CFD, and CFP), and to an agent of four of the fluoroquinolones group (EFX) (Table -8).
A molecular genetic study of the presence of resistance to antibacterial drug genotypes among isolated E. coli isolates identified genes encoding resistance to β-lactams in 15.4%, sulfonamides in 30.8%, and quinolones in 9.3% (Table -10).

Discussion
This study demonstrates the relevance of microbial contamination in retail food products.Three bacterial genera belonging to the Enterobacteriaceae family, Escherichia, Enterobacter, and Citrobacter, were identified in samples of different types of cheese produced in Kazakhstan in this study.The first two genera are most often isolated from raw milk [28], and Serratia is also often found in commercial cheeses [29,30].Although coliforms are considered to be thermolabile and cannot withstand pasteurization, 44.9% of the studied cheeses were found to be contaminated with them.At the same time, approximately 70% of the contaminated samples contained E. coli, which was identified in almost a third of the samples of cheeses of various types produced in the country, and these results are comparable with those of studies of cheeses and milk in other regions of the world [31,32].
It should be noted that, according to Kazakhstani standards, the microbiological veterinary and sanitary evaluation of dairy products is carried out only on the basis of the level of contamination with coliforms, whereas international standards suggest that such products should be examined in terms of the degree of E. coli contamination.Studies have shown that if assessed according to PHLs standards [33], the main proportion (92.3%) of domestic cheese contaminated with this species was acceptable, and 7.7% was unacceptable for consumption by CFU/g.At the same time, 4/5 of the products unacceptable for this indicator were soft cheeses.The detection rate of E. coli-contaminated cheese samples in the northern region was relatively higher (approximately 12) than that in the central and eastern regions.This species has not been found in cheeses from the southern region, probably due to the small number of samples studied in the region.In all geographical locations where E. coli was detected in the products, soft cheeses without vacuum packaging (100%) were the most contaminated.In addition, contamination of cheeses with bacteria was also correlated with the size of the enterprise.Therefore, the level of product contamination of small farms was more than twice that of large and medium-sized producers, and E. coli was isolated in 65% of the cheese samples sold.
At present, there is an increase in the global demand for farm cheeses, as consumers increasingly prefer distinctive products with regional flavor, characteristic tastes, and characteristics [34].As a means of income diversification, many farmers prefer manual small-scale handicraft production, and this cheese-making is becoming popular among the population of remote mountainous and steppe regions of Central Asia.However, as has been shown in the present studies, such production does not appear to comply with proper hygiene requirements, leading to high contamination of the final product with coliforms and other microflora.In some European countries, in particular France, up to 25.0% of samples of farm cheeses were positive for pathogenic STEC [35].
It should be noted that the pathogenic E. coli O157:H7 strain was detected for the first time in Central Asia from a sample of soft cheese from a small-scale producer in this study.The level of contamination of domestic cheese samples with this strain was 0.5%.In the past two decades of this century, endemic outbreaks of E. coli O157:H7 toxic infection among humans caused by cheese consumption have been documented in France [36], the USA [37], and Canada [38].Therefore, food contamination by STEC is the greatest concern to public health in most countries, and the Food Safety System (FSS)/Food Standard Agency (FSA) considers any STEC strain as potentially pathogenic [39].In all cases of E. coli O157:H7 outbreaks caused by cheese consumption, improper hygienic production conditions, such as lack of effective cleaning and disinfection procedures, lack of sanitary treatment, and insufficient cheese aging time, have been identified in enterprises where contaminated products were produced.Microorganisms can grow in biofilms in dairy production equipment, creating ideal conditions for the long-term survival of E. coli in the environment, which threatens the food safety of commercial products [40].
According to recent epidemiological studies, EPEC averaged 5.0% in the etiological structure of acute diarrhea in children in Kazakhstan; only serotypes 020, 0111, and 055 of this species have been described in the country [41].The first detection of E. coli O157:H7 in cheese suggests the possibility of contamination of dairy products with other STEC strains.To fully monitor the food safety of animal products sold, thoroughly studying the microbial landscape in relation to this E. coli pathogenic group is necessary.
The presence of antimicrobial-resistant bacteria in dairy products is recognized as a global problem of food safety and public health due to the potential transmission risk of resistant pathogens to the human population [18,19].The resistance of E. coli strains isolated from cheeses produced in Kazakhstan to antibacterial agents showed that bacterial isolates were resistant to 65% of the 20 drugs studied.The proportions of AMX, NA, and CFX-resistant isolates were 3%, 5%, 6%, 9%, 11%, 11%, 12%, 12%, 14%, 15%, 20%, and 31% for NFX, OFX, DOX, MPN, SFM+TMT, FD, FZ, LEV, TET, 20 and CFN, respectively.The data obtained are consistent with the results of many studies conducted in different countries, where it was found that resistance to sulfonamides, tetracyclines, and aminoglycosides, in general, is the most common among E. coli strains isolated from food [42].
It should be noted that the resistance of 16 isolated E. coli isolates (25% of the total number studied) was multi-resistance because they were resistant to three or more classes of antimicrobial drugs.Similar results have been reported in many previous studies, where E. coli strains with multidrug resistance have been detected in food and environment [13,[43][44][45].
Thus, E. coli strains resistant to antimicrobial drugs are widespread in the production environment of dairy enterprises in Kazakhstan and can seriously threaten public health because such bacteria can carry and transmit genes encoding determinants of antibiotic resistance [46].Molecular studies of E. coli isolates in the framework of this work confirmed the presence of eaeA virulence and resistance genes to β-lactam, sulfonamide, and quinolone groups.These genes are often associated with mobile genetic elements, such as plasmids, bacteriophages, and transposons, which can be exchanged between bacteria belonging to different strains and phylogenetic lines and create new combinations of virulence and resistance factors [43].As a result, virulent and antibacterial-resistant bacteria acting as a reservoir can transmit these genes to commensal and pathogenic microorganisms inside the human digestive tract [45] and increase the incidence of foodborne diseases [39,47,48].
Thus, the results of these studies indicate that in Kazakhstan, there are potential risks for increasing the antibiotic resistance of the intestinal microbiome, as well as endemic outbreaks of food infections and intoxication of the human population associated with contamination by E. coli in cheeses produced in the country.Therefore, to supplement the regulatory documents of Kazakhstan on monitoring and control of food coliform enteropathogens, we consider it relevant to further comprehensive research on domestic and imported dairy products' contamination with E. coli.This investigation will be of great scientific and practical importance for dairy businesses and veterinary and medical services in the context of the One Health concept and to ensure food safety in the country.

Conclusion
E. coli contamination was found in 31.4% of commercial Kazakh cheese samples of different brands, regardless of the geographical location and capacity of dairy enterprises.According to microbiological indicators, soft cheeses (65% of samples) from small farmers (80%) packaged at retail locations (100%) were the most contaminated with E. coli.For the first time in Central Asia, the enteropathogenic strain E. coli O157:H7 was detected in 0.5% of cheese samples.E. coli isolates from cheese samples were resistant to 65% of the tested antibacterial drugs and contained resistance genes to β-lactams, sulfonamides, and quinolones groups.These results confirm the need to develop effective mechanisms to control the production and sale of ready-to-eat dairy products in the event of contamination by E. coli and its pathogenic and multi-resistant antibacterial drug strains.

Figure- 1 :
Figure-1: Geographical locations of sampling (red marking shows the area from where the samples were collected).

Table- 6 :
Dependence of the cheese contamination by Escherichia coli on the enterprises' size.

Table - 1
: Sampling of products under study (by type of cheese).

Table - 2
: Primers used to confirm the presence of virulence determinants in the multiplex PCR.

Table - 4
: Cycling conditions of the primers during PCR.

Table - 5
: Microbiological sanitary assessment of Escherichia coli contaminated cheese samples, according to PHLs.

Table - 3
: Primers used for antibiotic resistance genes and virulence detection.

Table - 7
: Contamination of cheeses with Escherichia coli depending on the packaging method.

Table - 10:
Genes for antibiotic resistance in cheese samples (n = 65) of Kazakhstani producers.