Introduction

Antibiotics are a class of antimicrobial agents that can combat bacteria and typically used in medicine and agriculture. Globally, antibiotics are the most popular and efficient drugs for treating bacterial infections, the incapacity of these pathogens to be treated is known as antibiotic resistance1.

Antibiotic or antimicrobial resistance is defined as the ability to withstand the medications intended to treat them. Thus, treatment-resistant illnesses are the result of these germs. Nowadays, antibiotic resistance is a significant health concern worldwide that contributes to increased mortality and morbidity, longer hospital stays, and higher medical expenses2.

Bacterial antibiotic resistance poses a serious threat to human, animal, and environmental health3. The treatment choices and effectiveness are restricted when these bacteria are more prevalent. Consequently, harmful bacteria that develop resistance to antibiotics are a serious threat to both human and animal health4.

Extended spectrum β-lactamases (ESBLs) are gram-negative bacteria belonging to the Enterobacterales family. ESBLs are enzymes produced by certain bacteria, including Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae), which render them resistant to many commonly used antibiotics5.

These bacteria can cause serious infections such as urinary tract infections (UTIs), bloodstream infections, and pneumonia. The World Health Organization (WHO) correctly considers ESBLs to be among the most difficult diseases6. Bacteria with β-lactam-resistant ESBL are one of the primary causes of concern. Since ESBLs are enzymes that confer resistance to several beta-lactam antibiotics, inactivation of multiple antimicrobial types, such as monobactams, cephalosporins, penicillin, and β-lactamases, complicates the treatment of infections5.

Most ESBL-encoding genes are found in transposon or plasmid insertion sequences. As a result, they have the potential to spread swiftly and cause resistance to other antimicrobials such as tetracyclines, aminoglycosides, trimethoprim, sulfonamides, fluoroquinolones, and chloramphenicol7,8. In both human and animal populations, the misuse or overuse of antibiotics results in the formation of drug-resistant bacteria through gene changes or horizontal spread of resistance genes by plasmids9.

According to reports from around the world, ESBL-producing Enterobacterales organisms or ESBL-E is found in animal and human commensals, environmental contaminants, and various biological niches. Therefore, animal production has been a major concern in recent decades because of its tight linkages to the food chain and its ability to serve as reservoirs, vehicles, or routes of transmission and dissemination of ESBL-E10.

Many factors contribute to antibiotic resistance, such as the use of antibiotics in agriculture, the way people ingest antibiotics, and environmental contamination by antimicrobial chemicals. The increase in the demand for antibiotics and their widespread and negligent use are major contributors to the development of resistant strains. One of the main causes of antibiotic-resistant bacteria in food animals is the ongoing use of antibiotics in their production11. Monitoring the spread of resistant bacteria from animals to humans at each step of the transmission pathway is crucial, because certain antibiotic classes are used in both humans and animals12.

As a result, ESBL-producing bacteria can be directly transferred to humans through food products or the oral fecal route from infested animals that eat raw foods13. However, the presence of enteropathogenic bacteria leads to an increase in consumer health problems, and fruits consumed by the skin, such as pears, apples, blackberries, grapes, and strawberries, are more likely to have these bacteria on their surfaces14.

From overall, monitoring antibiotic-resistant bacteria and genes in human and food materials is necessary in settings where humans have routine contact with agriculture and livestock products because of the increased risk of spread of resistance genes in these interactions.

For Southeast Asian (SEA) review study has documented a rise in multidrug resistance in Gram-negative bacteria, particularly ESBL and carbapenem resistance in Enterobacterales15. In addition, in order to address the growing antimicrobial resistance (AMR) in the setting, it is necessary to obtain a comprehensive assessment of the available evidence in order to guide future policies and initiatives, particularly with regard to monitoring and surveillance.

According to previous studies reported, the genes that encode these enzymes are commonly located on mobile genetic elements such plasmids and transposons, which can promote horizontal transmission of resistance among Enterobacterales16,17. And due to the outbreak of the E. coli ST131 lineage in SEA, ESBLs type blaCTX-M have become more and more isolated18. AMR gene levels were also found to be high in SEA K. pneumoniae isolates, underscoring the significance of genomics-based surveillance, which can help standardize data for site comparison and detect regional and national variations19.

Nevertheless, reports of these issues are limited in Thailand. Therefore, the purpose of this study was to assess the potential contamination of ESBL-producing Enterobacterales antibiotic-resistant bacteria and ESBL genes in food materials collected from fresh markets in Chiang Mai province, Northern Thailand, to determine risk factors for contamination and prevent the transfer of resistant microbes from farms to people. Furthermore, ESBL genes in clinical specimens were determined and evaluated to understand the genetic diversity of antibiotic-resistant organisms that affect human health in this area.

Material and methods

Sample collection

Food material samples collection

This cross-sectional study collected food material samples from fresh markets in Chiang Mai Province, Northern Thailand. All raw food material samples, including chicken and pork meat, fruit, and vegetables, were randomly purchased and collected between September 2023 and February 2024. All sample types were random sampling for 20–50 samples. These samples were kept in clean plastic and zip lock bags, and then transported to the laboratory in a cold container with an ice pack as soon as possible or within eight hours to detect extended-spectrum β-lactamase (ESBL)-producing Enterobacterales antibiotic-resistant bacteria. The total number of food samples collected is listed in Table 1.

Table 1 Total numbers of food material samples collected from difference fresh markets in Chiang Mai, Thailand between September 2023–February 2024, n = 279.
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Residual of clinical cultured specimens collection

Ethics approval and consent to participate

This study was reviewed and approved by the Ethics Committee of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (Research ID. 0537 Study: NONE-256-0537) for the collection of clinical cultured specimen residuals which were categorized as exempted specimens. All study methods were carried out in accordance with relevant guidelines and regulations. In addition, due to the study did not directly involve patients, human material, or personal data identifiers therefore the patient informed consent was not obtained in this study. The need to obtain informed consent was waived by the Ethics Committee of the Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

Specimen collection

All bacterial isolates included in this study were collected from the microbiology laboratory in Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University, at the same time of food material samples collected. A total of 39 residual isolated colonies of ESBL-producing E. coli and ESBL-producing K. pneumoniae which cultured from different kinds of clinical specimens include blood, urine, pus, sputum and body fluids between September 2023–February 2024 (Table 2) was collected.

Table 2 The details of residual isolated colonies of ESBL-organisms collected from clinical cultured specimens between September 2023–February 2024.
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The isolated colonies were subjected to antimicrobial susceptibility tests using fully automated VITEK2, bioMérieux, and Sensititre Aris®2X, Thermo Scientific™ machines. The standardized antimicrobial sensitivity test for ESBL detection was confirmed, a double disk synergy test was performed using Muller Hinton Agar (MHA) media agar, and the results were interpreted according to the Standards for Antimicrobial Susceptibility of the Clinical Laboratory Standards Institute (CLSI) protocol20.

Briefly, presumptive ESBL-positive isolated colonies were suspended in broth culture at a McFarland standard of 0.5. A sterile swab was dipped into the standardized suspension of bacteria and streaked on the entire surface of the MHA plate. After Subsequently, ceftazidime (CAZ 30 µg), cefotaxime (CTX 30 µg), Ceftazidime/Clavulanic acid (CAC 30/10 µg), and cefotaxime/clavulanic acid (CEC 30/10 µg) were applied to the inoculated plate and incubated at 37 ± 2 °C for 18–24 h. The zones of inhibition were measured to the nearest millimeter at the back of the inverted culture plate to examine the antimicrobial drug susceptibility test, which was considered ESBL-positive when an increase of ≥ 5 mm in the zone diameter of any of the antibiotic disks was used towards the center21. While an increase < 5 mm in the zone diameter, the result was considered ESBL-negative. For quality control, E. coli ATCC® 25,922 and ATCC® 35,218 were used.

Microbial culture, identification and drug susceptibility test (DST) for ESBL-producing bacteria

All food material samples were tested for ESBL-producing organisms by microbial culture on selective media and confirmed using the DST test.

Bacterial culture test (culture-based method)

A sterile swab was used to swab the surface of each 100 g of raw food sample (one by one), which was then brought to the detection of ESBL-producing bacteria by streaking back and forth in a zigzag pattern on the surface of the CHROMID® ESBL selective media plate (bioMerieux SA, Marcy l’Etoile, France)22. Subsequently, these selective media plates were incubated at 35 ± 2 °C for 18–24 h. The processing procedures for Enterobacterales isolation and selective enrichment were based on previous studies23,24.

Bacterial isolation and identification test

The plates were visually inspected to determine whether the colony growth colors corresponded to the presumed Enterobacterales bacteria (pink or blue/turquoise colonies on CHROMID® ESBL agar). Suspected colonies were isolated on MacConkey agar plates. After incubation at 35 ± 2 °C for 18–24 h, the isolated single colony of suspected bacteria was identified as the organism strain using the automated matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS), VITEK® MS (bioMerieux, Marcy l’Etoile, France)22.

Antimicrobial resistant drug susceptibility test (DST)

Isolated colonies were identified as members of the order Enterobacterales that grew on the CHROMID® ESBL selective media plate (bioMerieux SA, Marcy l’Etoile, France) and were then confirmed to produce ESBL by antimicrobial-resistant DST. The standardized antimicrobial sensitivity test for ESBL detection was performed by a double-disk synergy test using Muller Hinton Agar (MHA) media agar, and the results were interpreted according to the Standards for Antimicrobial Susceptibility of the Clinical Laboratory Standards Institute (CLSI) protocol20. For quality control, E. coli ATCC® 25,922 and ATCC® 35,218 were used.

Detection of ESBL-antibiotic resistance genes

PCR amplification and nucleotide sequencing of ESBL genes were performed in these steps. The genomic DNA of all ESBL isolate colonies collected from food material and clinical specimens was extracted and amplified for ESBL-genes, including blaCTX-M and blaTEM, using specific primers followed these procedures.

Extraction of bacterial DNA

Plasmid DNA was extracted using the boiling method25. Briefly, the isolated bacterial colonies were resuspended in a 500 µL of sterile deionized water tube and then boiled at 100 °C for 10 min. Lysates were centrifuged at 12,000 × g for 10 min. The supernatant was collected and used as a DNA template. DNA quantity and purity were determined using NanoDrop One (Thermo Fisher Scientific™, Madison, WI, USA) at wavelengths of 260 and 280 nm.

Molecular identification of ESBL-genes by polymerase chain reaction (PCR)

Extracted bacterial DNA was amplified by PCR to detect antibiotic resistance genes using gene-specific primers listed in Table 3.

Table 3 The primers used for PCR amplification of ESBL genes.
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Briefly, the amplification reaction (25 µL) contained 13 µl of 2 × Taq PCR StarMix (Dye) (GenStar, China), 1 µL of 10 µM each forward and reverse primer, 10 µL of sterile deionized water, and 2 µL of extracted DNA (> 50 ng/µL). The thermal cycling conditions were 94 °C for 5 min, followed by 36 cycles of denaturation at 94 °C for 30 s, annealing at 63 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. Finally, PCR products were analyzed by gel electrophoresis to visualize the quality and expected size of plasmid DNA, using 1.5% (w/v) agarose gel and Super GelBlue™ (US EVERBRIGHT® INC., China) staining.

For quality control, known negative and positive samples for blaCTX-M and blaTEM amplification were used to perform along the procedure.

Interpretation of nucleotide sequences

The amplified DNA with representative of antibiotic drug resistance genes were sent to the company of Hunan Branch, Beijing Tsingke Biotech Co., Ltd. (China) to perform DNA sequencing tests. The nucleotide sequences were then examined using the Basic Local Alignments Search Tool (BLAST) software available at the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov) sequence databases28, which determines the statistical significance by comparing protein or nucleotide sequences to sequence databases and finds regions of similarity between biological sequences.

Results

Prevalence of ESBL-producing bacteria in food materials

In this study, raw food material samples were used to detect ESBL-producing organisms by culturing on CHROMID® ESBL selective media (bioMerieux SA, France). The presumptive ESBL-positive isolated colonies were visually inspected to determine whether the colony growth colors corresponded with the presumed Enterobacterales bacteria. The expected isolated colonies with pink or blue/turquoise colonies on CHROMID® ESBL agar (Fig. 1a and b) were identified as the microorganism strain using the automated MALDI-TOF MS, VITEK® MS (bioMerieux, Inc., Germany) machine. Finally, ESBL-positive isolated colonies were confirmed to be ESBL-producing organisms by a standardized antimicrobial sensitivity test for ESBL detection following the CLSI protocol 20,21.

Fig. 1
figure 1

The visually inspected colony growth colors corresponded with presumed Enterobacterales bacteria on selective CHROMID® ESBL agar plate (bioMerieux SA, France). (a) The result of pink color colonies on the upper part of CHROMID® ESBL agar (bioMerieux SA, France) demonstrated the ESBL-producing E. coli growth; (b) the result of blue/turquoise color colonies on the upper part of CHROMID® ESBL agar demonstrated the ESBL-producing K. pneumoniae growth. The lower part of both CHROMID® ESBL agar plates with no colony found demonstrated no ESBL-producing organism growth.

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From overall 279 raw food material samples; 43 pork and 33 chicken meats, 28 tomatoes, 33 cabbages, 30 lettuces, 27 grapes, 28 Chinese cabbage, 31 basils and 28 peppermints were collected during September 2023–February 2024 from fresh markets in Chiang Mai province, Thailand.

Of these raw food material samples, this study found that the prevalence rate of ESBL-producing organisms was 17.92% (50/279), including 15.41% (43/279), 2.15% (6/279), and 0.36% (1/279) for ESBL-producing E. coli, ESBL-producing K. pneumoniae and ESBL-producing K. oxytoca, respectively (Fig. 2).

Fig. 2
figure 2

The prevalence of ESBL-producing Enterobacterales bacteria detected on raw food material samples collected from fresh markets in Chiang Mai Province, Thailand (n = 279).

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Among the 50 ESBL-producing Enterobacterales bacteria contaminating the food samples, 86.00% (43/50), 12.00% (6/50), and 2.00% (1/50) were ESBL-producing E. coli, ESBL-producing K. pneumoniae and ESBL-producing K. oxytoca, respectively (Fig. 3a). Raw chicken meat samples had the highest detection of these organisms at 50% (25/50), followed by raw pork meat samples at 48% (24/50), and raw tomato at 2% (1/50) (Fig. 3b).

Fig. 3
figure 3

(a) The prevalence of ESBL-producing Enterobacterales bacteria detected in raw food material samples (n = 50). (b) The percentage of raw food material samples were ESBL-producing Enterobacterales bacteria detected. For basil, cabbage, Chinese cabbage, lettuce, peppermint and grape samples, ESBL-producing organisms were not detected.

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ESBL-producing Enterobacterales organisms were detected in 23 of 33 (69.70%) ESBL-producing E. coli and 2 of 33 (6.06%) ESBL-producing K. pneumoniae isolates obtained from chicken meat samples. While 20 of 43 (46.51%) were ESBL-producing E. coli, 4 of 43 (9.30%) were ESBL-producing K. pneumoniae obtained from pork meat samples, and 1 of 28 (3.57%) were ESBL-producing K. oxytoca obtained from tomato. No ESBL-producing organisms were detected in the other raw food material samples. The number of ESBL-producing organisms isolated from different raw food material samples is shown in Table 4.

Table 4 The prevalence of ESBL-producing Enterobacterales bacteria detected on each raw food material samples.
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Detection results of ESBL-antibiotic resistance genes

ESBL-antibiotic resistance genes in food material samples

From the 50 food material samples with ESBL-producing bacteria, 78 isolated colonies were obtained. Sixty-eight isolated colonies were obtained from ESBL-producing E. coli, 9 colonies were obtained from ESBL-producing K. pneumoniae and 1 colony was obtained from ESBL-producing K. oxytoca. All colonies were detected and confirmed for ESBL-antibiotic resistance genes, blaCTX-M and blaTEM, using molecular identification techniques with PCR and nucleotide sequencing.

The PCR products of ESBL genes detected by gel electrophoresis for the PCR amplification results of this study are shown in Fig. 4. Among the 78 extracted DNA samples of ESBL-producing E. coli, K. pneumoniae and K. oxytoca; 75 samples (75/78, 96.15%) provided positive PCR amplification results for blaCTX-M gene with 593 bp of PCR product on gel electrophoresis, and 34 samples (34/78, 43.59%) provided positive results for blaTEM gene with 445 bp of PCR product (Fig. 5).

Fig. 4
figure 4

The gel electrophoresis results of ESBL-producing genes detected by PCR amplification. The PCR products size 593 bp are represented for blaCTX-M gene positive samples whereas PCR products size 445 bp are represented for blaTEM gene positive samples. For blaCTX-M gene, sample no. S1-S4, S6-S7, S9-S11 were provided positive results whereas sample no. S5, S8 and S12 were provided negative results. For blaTEM gene, sample no. S1-S2, S5, S7, S9-S12 were provided positive results whereas sample no. S3-S4, S6 and S8 were provided negative results.

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Fig. 5
figure 5

The prevalence of ESBL-genes detected from 78 extracted DNA samples of ESBL-producing organism obtained from food materials samples.

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Moreover, for these 78 positive PCR amplification samples, the amplified DNA or PCR products were further subjected to DNA sequencing tests and nucleotide sequences were examined using the BLAST program to confirm the representative ESBL genes. Among these samples, for blaCTX-M gene; 100% (68/68), 66.67% (6/9) and 100% (1/1) were detected from ESBL-producing E. coli, K. pneumoniae and K. oxytoca, respectively. For blaTEM gene, 41.18% (28/68) were detected in ESBL-producing E. coli, 66.67% (6/9) were detected in ESBL-producing K. pneumoniae and no blaTEM gene was detected in ESBL-producing K. oxytoca. The overall results of the ESBL-producing genes detected by PCR Amplification and DNA sequencing are shown in Table 5.

Table 5 The prevalence of ESBL-genes detected in ESBL-producing organisms from food material samples by using PCR amplification and confirmed with nucleotide sequencing methods.
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The nucleotide sequences of the positive PCR amplification samples for blaCTX-M and blaTEM are shown in Figs. 6 and 7, respectively.

Fig. 6
figure 6

The nucleotide sequences of Sample no. S1 which positive for ESBL blaCTX-M gene by PCR amplification method. The sample results examined from BLAST software available at the National Center for Biotechnology Information (NCBI) website represented 99.27% identities with the E. coli strain CTX-M family extended-spectrum class A beta-lactamase (blaCTX-M) gene, partial cds from GenBank Sequence accession ID. OP491380.1 data.

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Fig. 7
figure 7

The nucleotide sequences of Sample no. S1 which positive for ESBL blaTEM gene by PCR amplification method. The sample results examined from BLAST software available at the National Center for Biotechnology Information (NCBI) website represented 100% identities with the E. coli strain TEM family beta-lactamase (blaTEM) gene, partial cds from GenBank Sequence accession ID. MN158355.1 data.

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ESBL-antibiotic resistance genes in clinical specimen samples

From a total of 39 isolated colonies of ESBL-producing organisms collected, 22 were obtained from ESBL-producing E. coli and 17 colonies were obtained from ESBL-producing K. pneumoniae. The determination and confirmation of ESBL-antibiotic resistance genes included blaCTX-M and blaTEM genes using molecular identification techniques with PCR and nucleotide sequencing by BLAST (blastn) software to search a nucleotide database, and the results are shown in Table 6.

Table 6 The prevalence of ESBL-genes detected in ESBL-producing organisms from clinical cultured specimens by using PCR amplification and confirmed with nucleotide sequencing methods.
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In this study, 37 of 39 samples (94.87%) were blaCTX-M genes, whereas 20 and 17 positive samples were from ESBL-producing E. coli and ESBL-producing K. pneumoniae, respectively. While blaTEM was detected in 26 of 39 samples (66.67%), 12 and 14 positive samples were from ESBL-producing E. coli and ESBL-producing K. pneumoniae, respectively.

ESBL-antibiotic resistance genes in food material samples and clinical specimen samples

In this study, the nucleotide sequences of ESBL genes detected in food material samples were further analyzed to evaluate the protein database using BLAST (blastx) software. Among the 75 blaCTX-M genes in raw food material samples, the results found blaCTX-M15 as 62.67% (47/75) and blaCTX-M14 in 37.33% (28/75). Among the 37 blaCTX-M genes in the clinical cultured specimens, blaCTX-M15 was 83.78% (31/37), blaCTX-M14 was 10.81% (4/37), and blaCTX-M27 was 5.41% (2/37). For overall blaTEM genes, the study results found 100% blaTEM-1 in both raw food material samples and clinical specimens (Fig. 8).

Fig. 8
figure 8

The prevalence of ESBL-genes detected in food material samples and clinical specimen samples by using PCR amplification and confirmed with nucleotide sequencing methods.

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Discussion

In this study, we assessed the rate of ESBL genes and antibiotic-resistant Enterobacterales bacterial contamination in food materials and further determined ESBL genes in clinical specimens to evaluate the relative abundance of antibiotic-resistant genes in these samples. The prevalence of ESBL-producing Enterobacterales contamination and ESBL-genes was investigated by gathering raw food, vegetables, fruits, and animal productsfrom fresh marketplaces. The majority of fruits and vegetables gathered for this study were typically raw, flesh-and-leaf-eaten foods. These study results showed that raw chicken meat samples had the highest prevalence rate of ESBL-producing organisms at 50%, followed by raw pork meat samples at 48%, and raw tomato at 2%, while no ESBL-producing organisms were found in other raw food material samples. Nevertheless, other pathogenic organisms were detected in these samples (data not shown)22, and almost all of them were opportunistic pathogens associated with various potential infections in immunocompromised and immunocompetent individuals.

Furthermore, we found that the total rate of ESBL-producing organisms was 17.92%, with high contamination of ESBL-producing E. coli (5.41%), ESBL-producing K. pneumoniae (2.15%), and ESBL-producing K. oxytoca (0.36%). Similar to other studies, antimicrobial organisms have also been detected in raw food materials, especially raw animal products, fruits, and vegetables29,30. Some studies have found that ESBL-producing bacteria, particularly E. coli and K. pneumoniae, can be found in various food products, especially those of animal origin (e.g., poultry, beef, pork), and contamination can occur during slaughtering, processing, or handling31. E. coli is the most prevalent host among gram-negative bacterial species from various families that carry ESBL genes, followed by K. pneumoniae11.

From previous reported, a study of Enterobacterales-order bacteria that produce ESBLs indicated that up to 18% of human community-onset infections may be caused by food products, with household transmission accounting for the majority of cases32. ESBL-producing Enterobacterales or ESBL-E can pose serious risks to food security and safety in both humans and animals. Animal and human populations can co-transmit ESBL-E through a variety of direct and indirect pathways, which has led to significant increases in commensal ESBL reservoirs in the environment. Because pathogenic bacteria in the environment can acquire ESBL genes from commensal bacteria, both humans and animals may be at a significant risk for health issues33.

Additionally, ESBL-producing bacteria have been detected in plants, soil, and water near cattle, markets, and agricultural settings34,35. Foodborne disease outbreaks have been associated with the consumption of fresh fruits and vegetables36. Several detrimental microorganisms have been linked to fruits and vegetables offered for sale in outdoor retail markets37. Owing to their collection from open fields and lack of washing prior to distribution for retail, food material samples are susceptible to microbial contamination. From farm harvest suppliers to selling locations, they may exhibit some mechanical or physiological damage, which could result in contamination by the time they are offered for sale in low-cost markets38. Therefore, the possible causes of fresh fruit contamination on farms, including biological soil amendments, human handlers, domestic animals, and irrigation water, must be considered.

In this study, to confirm the ESBL genes detected, we compared the nucleotide sequences to the sequence databases of the Basic Local Alignment Search Tool (BLAST) program. This tool was used to identify regions of similarity between the biological sequences28. In addition to identifying members of gene families, BLAST can be used to infer functional and evolutionary relationships between sequences. In our study, we found that the majority of the ESBL genes detected were those that generate enzymes of the CTX-M type, followed by the TEM type in both raw food material samples and clinical specimen samples. We detected 96.15% and 94.87% blaCTX-M genes and 43.49% and 66.67% blaTEM in raw food and clinical samples, respectively. No significantly different between the prevalence of resistance genes detected in these two groups of sample (p > 0.05, by Fisher’s Exact test). Moreover, in this study, we also found that the majority of ESBL genes detected in raw food and clinical samples were blaCTX-15 gene, followed by blaCTX-14 genes. The blaCTX-27 gene was detected in clinical samples but not in raw food samples. In this study, for raw food samples, the prevalence of blaCTX-M gene detected in ESBL-producing E. coli and K. oxytoca was higher than in ESBL-producing K. pneumoniae as 100% and 66.67%, respectively. Among the clinical samples, the prevalence of blaCTX-M detected in ESBL-producing K. pneumoniae was slightly higher than that in ESBL-producing E. coli at 100% and 90.91%, respectively. For blaTEM gene detected in both raw food and clinical samples, we found most of them were blaTEM-1 gene and the prevalence of gene detected in ESBL-producing K. pneumoniae was higher than in ESBL-producing E. coli. This result’s finding was similar to several studies that reported the frequency of blaTEM were high among Enterobacterales isolates from hospitalized patients due to this resistance gene related to the highly antibiotic used in this population39,40.

From previous study, the most common ESBL category has been found to be CTX-M-type enzymes, which have displaced TEM and SHV as the most common ESBL types41. Isolates containing CTX-M have been discovered in companion animals, food products, livestock, the environment, and nosocomial and community environments42. And most CTX-M gene isolates containing the blaCTX-M-14, blaCTX-M-15 and blaCTX-M-27 genes43,44,45. In addition to hospitals and communities, over 80 different types of CTX-M have been found in fresh vegetables, water, animals, and the environment46. CTX-M variations have been reported in P. aeruginosa, Acinetobacter spp., and various members of the order Enterobacterales, including E. coli and K. pneumoniae47,48. Furthermore, Shigella species and S. enterica serovar Typhimurium have a high prevalence of CTX-M-type ESBL49.

It is clear that various antibiotic regimes cause genotypic ESBL-producing E. coli to frequently differ among nations and regions50. Different genes were seen to coexist within the same isolation; the most prevalent genotypic pattern was the combination of blaTEM and blaCTX-M. This may result in problems with enzyme combinations. Additionally, multi-blaCTX-M isolates may result in more tenacious infections50. Antibiotic resistance genes can spread from one cell to another by conjugation, transformation, or transduction and are commonly found on plasmids or transposons. The resistance can be swiftly acquired by bacteria of different species through gene exchange. Horizontal transmission is the term for this procedure. For doctors treating infectious disorders, this kind of drug resistance poses serious challenges51.

In Thailand, it has been shown that the antimicrobial stewardship program (ASP) improves clinical outcomes, decreases antimicrobial consumption, encourages appropriate antimicrobial use, and slows the emergence of AMR without affecting patient outcomes in terms of infection-related morbidity and mortality52. Therefore, from overall study’s finding results focused on the detection of antibiotic-resistant ESBL organisms and genes in raw food materials and clinical samples. We found similar ESBL-producing organisms and genes in both the sample types. Inadequate agricultural and livestock sanitation and hygiene standards, bacteria with resistant genetic components can migrate from farms to the surrounding environment by contaminating the soil and water near the farm with livestock dung. Therefore, acknowledging ESBL antibiotic resistance in food materials requires a multifaceted approach involving monitoring, public education, regulatory oversight, and research.

Nevertheless, to improve the study quality in the future, to decrease any limitation of the study must be concerned. Regarding to DNA extraction and purification technique, using the commercial kit in experiment will provided high DNA quality and yield. For the limitation of the small number of sample collected in the study area, due to this study was a preliminary study to detect antibiotic resistance organisms and genes in our setting, the expanding sample types and study area should be more evaluated further. The continued research is needed to evaluate the prevalence of antibiotic-resistant microorganisms contaminating other food material samples. Finally, the laboratory and hospital settings, routine screening and prompt identification of ESBL-producing bacteria are essential for infection management and treatment strategies to prevent outbreaks.

Conclusion

In this study, ESBL-producing Enterobacterales resistance organisms and genes were detected in food material and clinical samples. The study’s findings on antibiotic-resistant organisms can be used to acknowledge the people involved in the food chain as collecting, slaughtering, processing, transporting, and storing raw food materials. As a result, people who handle raw meats and vegetables should be awareness and concerned about food safety and hygiene. It is necessary to be concerned about the possibility of acquiring illnesses from antibiotic-resistant bacteria and opportunistic pathogens when handling or consuming these foods. In addition, they should be mindful of the dangers and risks of infections caused by opportunistic and antibiotic-resistant pathogens. Therefore, it is crucial to enhance food safety and protect public health from the threats posed by antibiotic-resistant bacteria. Educating consumers about safe food handling and cooking practices can help to reduce the risk of exposure. Awareness campaigns can inform people about the risk of antibiotic misuse in agriculture. Policymakers may need to consider stricter regulations on antibiotic use in livestock to mitigate the development of antibiotic resistance. Development plans for the control and surveillance of antibiotic resistance of pathogens and the use of antibiotic drugs for the future treatment of bacterial infections that support prevention, healthcare, and public health systems are needed.