High prevalence of class 1 integron, biofilm formation, and antimicrobial resistance profiles of Escherichia coli isolated from cattle, water, and soil in Bangladesh

Abstract

Background

Antimicrobial resistance (AMR) is a global threat. Escherichia coli is an important reservoir of resistance genes, and the coexistence of mcr with class 1 integron (intI1) is alarming, mcr confers resistance to colistin, a last-resort antibiotic, while intI1 often carries multiple transferable resistance determinants. Although colistin use in livestock is banned in Bangladesh, data on colistin-resistant E. coli in food animals, particularly cattle, remain scarce. This study investigated multidrug-resistance patterns, biofilm formation, and the prevalence of intI1 and its association with mcr and other resistance determinants in E. coli from Bangladeshi cattle.

Methods

A total of 254 samples (feces, rectal swabs, soil, and water) were collected from cattle farms across three divisions of Bangladesh (Dhaka, Sylhet, and Barisal). E. coli isolates were identified using culture methods and confirmed with the VITEK-2 system and PCR. Antimicrobial susceptibility testing was performed using VITEK-2 system following CLSI guidelines. The presence of class 1 integron and mcr (mcr-1 to mcr-5) genes was detected by PCR. Biofilm formation was assessed using the crystal violet assay. Statistical analysis was conducted in R using Chi-square exact test, with significance set at p < 0.05.

Results

E. coli was detected in 76.4% of samples, with the highest prevalence in feces (86.8%). Antimicrobial testing revealed complete sensitivity to several antibiotics, but resistance occurred to cefuroxime (33%), ciprofloxacin (26.8%), and amoxicillin-clavulanic acid (22.2%), with all isolates showing intermediate response to colistin. Overall, 29.4% of isolates were MDR, though no XDR or PDR were detected. Class 1 integron was present in 52.6% and the mcr-1 gene in 21.7%, both significantly associated with MDR (p < 0.001). Water isolates showed the highest proportion of strong biofilm producers (25.5%).

Conclusion

This study reveals a high prevalence of class 1 integron and the mcr-1 gene in MDR E. coli from food animals in Bangladesh, highlighting their role in resistance dissemination and the public health risk of colistin resistance. The strong association of integron with MDR and mcr-1 underscores the need for prudent antibiotic use, routine surveillance, and stronger antimicrobial stewardship in livestock to limit the spread of resistance to humans.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-026-04786-2.

Introduction

Antimicrobial resistance (AMR) has emerged as one of the most critical global public health threats of the 21st century [1]. The widespread use and misuse of antimicrobials in humans, animals, and agriculture have accelerated the development of MDR bacteria, posing serious challenges to the treatment of infectious diseases [2]. Among the many bacterial species implicated in AMR, E. coli, a commensal inhabitant of the intestinal tract of humans and animals, occupies a central role [1, 3]. While many E. coli strains are harmless, pathogenic and resistant strains represent a major public health concern due to their ability to cause severe diseases and their high potential for horizontal gene transfer [4]. The rapid emergence and dissemination of MDR E. coli in both clinical and non-clinical settings underscore the urgent need to understand the mechanisms underlying their resistance [4, 5].

In the context of food animals, the emergence of MDR E. coli carrying integron is particularly concerning [6]. Food-producing animals are frequently exposed to antimicrobials, not only for therapeutic purposes but also for growth promotion and prophylaxis [7]. Such practices, especially when implemented without strict regulation, create selective pressure that favors the persistence and spread of resistant strains [7, 8]. These resistant bacteria can be transmitted to humans through direct animal contact, consumption of contaminated food products, or environmental pathways [9]. This zoonotic transfer represents a serious risk to public health, as it reduces the effectiveness of essential antimicrobials in human medicine [10]. Food animals have been recognized as reservoirs of resistance determinants, including class 1 integron, especially in low- and middle-income countries where antimicrobial use in animal husbandry is frequently poorly regulated [2, 10]. Class 1 integron are the most prevalent and clinically significant among the integron classes, frequently carrying genes conferring resistance to multiple antibiotic classes [11]. Therefore, this study focused on class 1 integron, as they play a major role in the dissemination of antimicrobial resistance, whereas other integron classes are less common and less well-characterized in clinical and environmental isolates [11, 12]. In addition, animal-derived waste can introduce antimicrobial-resistant bacteria and resistance genes into surrounding soil and water systems through manure application, runoff, and improper waste disposal [13, 14]. These environmental compartments may act as long-term reservoirs and transmission pathways for integron-bearing MDR E. coli [15], facilitating their persistence, dissemination, and potential re-entry into the food chain and human populations [16].

Integron is the genetic platforms that capture, integrate, and express gene cassettes, many of which encode antimicrobial resistance determinants [12, 17]. They are not self-mobile but are often located on plasmids and transposons, which facilitate horizontal gene transfer among bacteria [18]. Among different classes, class 1 integron is the most prevalent and have been strongly associated with resistance to multiple classes of antimicrobials, including aminoglycosides, β-lactams, sulfonamides, and tetracyclines [18, 19].

Bangladesh, like many developing countries, is experiencing a rapid intensification of livestock and poultry farming to meet the growing demand for animal protein [20]. However, antimicrobial use in animal production remains largely uncontrolled, with antimicrobials frequently administered without prescription or veterinary oversight [21]. Several studies have reported a high prevalence of MDR E. coli in poultry, cattle, and companion animals in Bangladesh [22–24]. Nevertheless, there is limited information on particular the role of integron. Understanding the prevalence of class 1 integron in MDR E. coli isolated from food animals in Bangladesh is therefore critical for identifying the genetic basis of resistance and for designing effective control strategies [25].

The detection of colistin resistance in cattle is particularly alarming, as colistin is considered a last-resort antibiotic for treating MDR infections and colistin use in livestock is banned in Bangladesh [1, 26]. The presence of colistin-resistant E. coli in food animals not only poses a risk to animal health but also raises serious public health concerns, given the potential for transmission of resistant strains or resistance genes through the food chain and the environment [27, 28]. This highlights the urgent need for continuous surveillance and strict antimicrobial stewardship to prevent further dissemination of colistin resistance in Bangladesh.

Previous research in other regions has demonstrated that class 1 integron are widespread among MDR E. coli from animals, and their prevalence often correlates with the intensity of antimicrobial use [29]. For example, studies in China and India have reported high frequencies of integron-positive isolates in food animal populations, highlighting the global dimension of this problem [29, 30]. However, given the unique farming practices, antibiotic use patterns, and environmental conditions in Bangladesh, the epidemiology of integron may differ and requires local investigation [31].

This study aimed to address this gap by determining the prevalence of class 1 integron and mcr genes in MDR E. coli isolated from food animals in Bangladesh. By identifying the determinants associated with the presence of class 1 integron, the findings of this research will provide insights into the potential for horizontal gene transfer of resistance traits within bacterial populations in animals and possibly to humans. Furthermore, the study will contribute to the growing body of evidence required to inform antimicrobial stewardship programs and policy decisions regarding antimicrobial use in food animal production in Bangladesh.

Materials and methods

Study area and sample collection

In total, 254 samples were obtained, consisting of 152 feces, 40 rectal swab, and 62 environmental samples (31 soil and 31 water). These were collected from three administrative divisions of the country: Dhaka (feces = 80, rectal swab = 16, soil = 11, water = 11), Sylhet (feces = 40, rectal swab = 12, soil = 10, water = 10), and Barisal (feces = 32, rectal swab = 12, soil = 10, water = 10). The samples originated from cattle farms, including both animal sources (feces) and surrounding environmental components (soil and water). The samples were collected aseptically following the protocol of Rahman et al. 2025 [22]. Following collection of samples, they were transported immediately to the Antimicrobial Resistance Reference Laboratory of the Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka-1341, Bangladesh with maintaining proper cool chain.

Before collecting samples, all required equipment was carefully prepared and sterilized following internationally recognized guidelines (ISO/TS 21872-1:2023) [32]. Buffered peptone water (Oxoid, UK) served as the transport medium for the samples. To ensure their integrity, the specimens were handled under a maintained cold chain, transported with care, and properly documented. Upon arrival at the laboratory, they were inoculated into nutrient broth and incubated at 37 °C for 24 h to promote bacterial growth. All laboratory procedures and analyses were conducted following established clinical techniques and validated methodologies [22, 33, 34]. Bacterial isolates were maintained by mixing cultures with a cryoprotectant composed of 1 mL Luria–Bertani (LB) broth and 0.5 mL of sterile 80% glycerol. The prepared stocks were divided into aliquots and stored at − 80 °C, in accordance with standard procedures for long-term preservation of bacteria in glycerol concentrations above 15% for future analyses.

Isolation and identification of E.coli

The samples were inoculated into nutrient broth and incubated at 37 °C for 24 h to promote bacterial growth. E. coli isolates were initially characterized phenotypically by examining their colony morphology, including shape, size, color, and edge features, following guidelines from the Manual of Methods for General Bacteriology [35]. On EMB agar, E. coli typically produced colonies with dark centers and a distinctive metallic green sheen, resulting from the interaction between eosin and methylene blue and active lactose fermentation, which lowers the pH of the medium. Confirmation of E. coli was subsequently performed using the VITEK^®2 system (bioMerieux, USA), which identified isolates based on their specific biochemical characteristics and PCR.

Antibiotic Susceptibility Testing (AST)

Antibiotic susceptibility testing was performed using the VITEK^®2 Compact system (bioMerieux, USA), and this system follows the Clinical and Laboratory Standards Institute (CLSI, 2020) guidelines [36, 37]. In brief, a bacterial suspension was prepared to match the 0.5 McFarland standard. Antibiotic susceptibility testing was carried out using commercially available disks, which included Amikacin (30 µg), Gentamicin (10 µg), Ertapenem (10 µg), Meropenem (10 µg), Imipenem (10 µg), Cefuroxime (30 µg), Cefuroxime Axetil (30 µg), Ceftriaxone (30 µg), Cefoperazone-Sulbactam (30 µg), Cefepime (5 µg), Cefoxitin (30 µg), Ceftazidime (30 µg), Ciprofloxacin (5 µg), Trimethoprim-Sulfamethoxazole (1.25/23.75 µg), Tigecycline (15 µg), Aztreonam (30 µg), Piperacillin (100 µg), Amoxicillin-Clavulanate (20/10 µg), Fosfomycin (200 µg), and Colistin (10 µg). E. coli ATCC 25,922 was utilized as the quality control strain.

Classification of bacterial isolates based on resistance profiles

Isolates resistant to three or more different classes of antibiotics were classified as MDR. Isolates that were susceptible to only one or two antibiotic classes were considered extensively drug-resistant (XDR), while those showing resistance to all tested antibiotic classes were designated as pan-drug resistant (PDR) [22, 38].

DNA extraction and molecular detection of class-1 integron & mcr-genes

The boiling method is a quick and simple approach widely used for extracting bacterial DNA. In this study, 1500 µL of an overnight E. coli culture, incubated at 37 °C in nutrient broth, was transferred to an Eppendorf tube. The sample was centrifuged at 12,000 rpm for 3 min to collect the bacterial cells as a pellet. The supernatant was discarded, and the pellet was then resuspended in 200 µL of sterile distilled water, followed by thorough vertexing to achieve complete homogenization [39, 40]. Additionally, the quality and concentration of the extracted DNA were assessed using a NanoDrop spectrophotometer.

The resuspended bacterial cells in the Eppendorf tube were subjected to a water bath at 95–100 °C for 5–10 min. This boiling process caused cell lysis, releasing genomic DNA into the solution. Immediately afterward, the tubes were placed on ice for 2–5 min to stabilize the DNA and minimize degradation. The samples were then centrifuged at 12,000 rpm for 5 min to pellet the cellular debris, separating it from the DNA-containing supernatant. The clear supernatant, containing the extracted DNA, was carefully transferred to a new Eppendorf tube without disturbing the pellet. The extracted DNA was stored at -20 °C for long-term preservation and further analyses. To confirm the E. coli, and to detect the colistin resistant genes (mcr1-mcr5) and class 1 integron (intI1), specific primers were used for PCR amplification. The details of the primers and PCR conditions are provided in supplementary table (TableS1).

Biofilm assay

The procedure started by streaking the isolates onto Nutrient Agar, followed by overnight incubation at 37 °C. A single colony from the plate was then inoculated into 5 ml of Nutrient Broth and incubated for 24 h at the same temperature. Subsequently, 500 µl of the bacterial culture was added to each well of a 56-well microtiter plate and incubated at 37 °C for 16–24 h. After incubation, the culture was gently removed, and the wells were rinsed 2–3 times with phosphate-buffered saline (PBS). To stain the biofilm, 500 µl of 0.2% (w/v) crystal violet solution was added to each well and left at room temperature for 10–15 min. The dye was then discarded, and the wells were washed again 2–3 times with PBS to remove excess stain, ensuring accurate measurements. The biofilm-forming capacity of the isolates was then classified into strong, moderate, weak, or non-biofilm formers, following the criteria of a previously validated study [41].

Statistical analysis

All datasets were organized in Microsoft Excel (MS-2021), and subsequent statistical analyses were conducted using R (RStudio, version 2024.12.1 + 563). To visualize the relationships between antibiotics and samples based on antimicrobial susceptibility test (AST) results, a heatmap was generated using the ‘pheatmap’ package in R. Chi-square test was applied to evaluate associations between the presence of class 1 integron and different variables, as it is appropriate for small sample sizes and non-normally distributed data. Statistical significance was defined as a p-value below 0.05.

Ethics statement

In order to respect ethical norms and guarantee the welfare of all participants and biological materials used, this study was conducted in complete accordance with the ARRIVE criteria. The Bangladesh Livestock Research Institute’s (BLRI) Technical Review Committee approved the research protocol (No. AHRD01/2022-23) before to its start, attesting to its compliance with national and international ethical standards. To reduce risk and protect anonymity, every step of the study from sample collection and processing to laboratory analysis was carried out with extreme caution. By encouraging openness, accountability, and respect for moral research methods, the study team stayed dedicated to moral behavior throughout the entire process.

Results

Prevalence of E. coli in food animals

The study shows a high overall prevalence of E. coli 76.37% (194 out of 254) in cattle and their environment. The highest occurrence was detected in feces samples 86.84% (132 out of 152), followed by rectal swabs 70% (28 out of 40). Environmental sources such as soil 56.25% (18 out of 32) and water 53% (16 out of 32) also showed notable contamination, indicating widespread presence of E. coli in both animals and their surroundings (Fig. 1).

Fig. 1.

Prevalence of E. coli

Resistant pattern from antimicrobial susceptibility testing (AST)

The antimicrobial susceptibility testing of E. coli isolates from cattle revealed a varied resistance pattern. The isolates showed complete susceptibility to amikacin, ertapenem, ceftazidime, cefepime, piperacillin, and fosfomycin, with 100% sensitivity. High susceptibility was also observed to gentamicin 95.9%, meropenem 92.3%, tigecycline 96.4%, and trimethoprim-sulfamethoxazole 85.6%. However, notable resistance was recorded against cefuroxime 33%, ciprofloxacin 26.8%, and amoxicillin-clavulanic acid 22.2%, with intermediate responses also present for meropenem, imipenem, ciprofloxacin, and amoxicillin-clavulanic acid. Alarmingly, all isolates showed an intermediate response to colistin, indicating possible emerging resistance. Overall, while most isolates remained susceptible to several critically important antibiotics, the observed resistance to fluoroquinolones, β-lactams, and colistin raises concern for potential therapeutic challenges and public health risks (Fig. 2). The heatmap of correlations revealed strong associations among the resistance patterns of different antibiotics, such as cefuroxime (CXM) and amoxicillin–clavulanic acid (AMC), as well as gentamicin (GM) and meropenem (MEM), which were highly correlated due to similar resistance patterns (Fig. 3).

Fig. 2.

Resistant patterns of the isolates

Fig. 3.

Heatmap with correlations

MDR and XDR E. coli from the isolates

The study found that 29.39% (n = 57) of E. coli isolates were MDR, while no isolates XDR or PDR. Among sample sources, MDR prevalence was highest in water samples (56.3%), followed by rectal swabs (35.7%), feces (25.8%), and soil (22%). The absence of XDR and PDR isolates indicates that although resistance to multiple antibiotic classes is already established, the bacteria have not yet progressed to extreme or pan-resistance (Fig. 4). Comparison between total resistant isolates and resistant isolates with intl gene are given at (Fig. 5).

Fig. 4.

Prevalence of MDR, XDR, and PDR among isolates

Fig. 5.

Comparison between total resistant isolates and resistant isolates with intl gene

Presence of class 1 integron and mcr genes

Overall, class 1 integron were identified in 52.57% of the samples, indicating a high prevalence of genetic elements associated with multidrug resistance. The mcr-1 gene, which confers resistance to colistin, was also detected in 21.65% of samples, highlighting a potential public health threat, as colistin is considered a last-resort antibiotic. Distribution across sample types showed similar patterns, with class 1 integron most frequently found in feces (53.8%) and rectal swabs (53.6%), and the mcr-1 gene highest in rectal swabs (25%). Notably, no plasmids or other mcr variants (mcr-2 to mcr-5) were detected. These findings suggest the circulation of mobile genetic elements that could facilitate the spread of resistance, particularly colistin resistance, within food animals and their surrounding environment (Fig. 6).

Fig. 6.

Prevalence of integron and mcr genes among selected isolates

Co-existence of class 1 integron and mcr-1

Among the 53 E. coli isolates carrying both class 1 integron and the mcr-1 gene, resistance was observed against a number of antibiotics. Notably, 38.5% of isolates were resistant to ciprofloxacin, 32.3% to ceftriaxone, 30.2% to amoxicillin-clavulanic acid, and 21.4% to trimethoprim- sulfamethoxazole. These findings suggest that while co-existence of class 1 integron and mcr-1 is concerning (Table S2).

Biofilm production

The study demonstrated that E. coli isolates from different sources varied in their ability to produce biofilms. In fecal samples, 24.2% were strong biofilm formers, while the majority were weak (40.9%) or non-biofilm producers (25.8%). Soil isolates were predominantly weak biofilm formers (61.1%), with only 11.1% showing strong biofilm formation. Water isolates showed the highest proportion of strong biofilm producers (25.5%), although weak (37.5%) and non-biofilm formers (25%) were also common. Rectal swab samples showed 21.4% strong and 17.9% moderate biofilm producers, while the largest proportion (42.9%) were weak formers. Overall, the findings indicate that a substantial proportion of E. coli isolates, particularly from feces, water, and rectal swabs, possess biofilm-forming ability, which may enhance their persistence and resistance in animal and environmental sources (Fig. 7).

Fig. 7.

Biofilm formation

Association of class 1 integron with mcr-1 and other factors

The bivariate analysis (Chi-square test) examined the association between class 1 integron and several factors (Table 1). No significant associations were observed with location, residence, sample type, or biofilm production (p > 0.05), indicating that the distribution of integron was relatively uniform across these categories. However, a strong and statistically significant association was found between class 1 integron and MDR (p < 0.001), with 75.4% of MDR isolates carrying class 1 integron compared to only 43.1% of non-MDR isolates. Similarly, the presence of the mcr-1 gene showed a significant association (p < 0.001), as 81.0% of mcr-1 -positive isolates harbored class 1 integron, compared to only 44.7% among mcr-1 -negative isolates. These findings suggest that class 1 integron are strongly linked with both MDR phenotypes and the presence of mcr-1, highlighting their potential role in the co-selection and dissemination of critical resistance determinants in cattle isolates.

Table 1.

Bivariate analysis (Chi-square test) of the association between the presence of class 1 integron and the mcr-1 gene, along with other factors

Variables	Categories	Presence of class 1 integron		Bivariate Analysis
		No (n %)	Yes (n %)	χ²	P value
Location	Dhaka	54 (45.0%)	66 (55.0%)	4.082	0.130
	Barisal	16 (66.7%)	8 (33.3%)
	Sylhet	22 (44.0%)	28 (56.0%)
Residence	Urban	44 (50.0%)	44 (50.0%)	0.429	0.512
	Rural	48 (45.3%)	58 (54.7%)
Sample type	Feces	61 (46.2%)	71 (53.8%)	0.609	0.894
	Soil	10 (55.6%)	8 (44.4%)
	Water	8 (50.0%)	8 (50.0%)
	Rectal swab	13 (46.4%)	15 (53.6%)
MDR	Yes	14 (24.6%)	43 (75.4%)	16.919	< 0.001
	No	78 (56.9%)	59 (43.1%)
mcr-1	Absent	84 (55.3%)	68 (44.7%)	17.310	< 0.001
	Present	8 (19.0%)	34 (81.0%)
Biofilm production	Strong biofilm‑former	18 (40.9%)	26 (59.1%)	2.244	0.523
	Moderate biofilm‑former	8 (38.1%)	13 (61.9%)
	Weak biofilm‑former	43 (51.8%)	40 (48.2%)
	Non‑biofilm‑former	23 (50.0%)	23 (50.0%)

Bold p-values indicate significantly associated variables

Discussion

This study revealed a high prevalence of E. coli (76.37%) in cattle and their surrounding environments in Bangladesh, with the highest occurrence in fecal samples (86.84%), followed by rectal swabs (70%). Similar high prevalence rates have been reported in previous studies from other low- and middle-income countries (LMICs), where close human–animal interactions, poor farm hygiene, and contamination of the environment facilitate the widespread circulation of E. coli [42, 43]. The substantial detection of E. coli in soil (56.25%) and water (53%) in our study further supports the evidence that livestock environments serve as important reservoirs of enteric bacteria, often linked to inadequate manure management and contamination of water sources used in farming practices [42].

The AST results demonstrated considerable variability, with isolates showing complete susceptibility to several critically important antibiotics, such as amikacin, ertapenem, ceftazidime, cefepime, piperacillin, and fosfomycin. This is consistent with studies from low- and middle-income countries, where aminoglycosides and carbapenems have likewise remained largely effective against E. coli isolates from food animals [44, 45]. This unusual pattern of susceptibility to ertapenem may be explained by differences in antibiotic uptake, efflux pump activity, or specific carbapenemase enzymes with varying substrate profiles [46, 47]. However, resistance to cefuroxime (33%), ciprofloxacin (26.8%), and amoxicillin-clavulanic acid (22.2%) was notable. Such resistance patterns have been frequently observed in South Asia, where third-generation cephalosporins and fluoroquinolones are commonly used in veterinary practice and livestock production, thereby exerting selective pressure [48]. Alarmingly, all isolates in our study displayed an intermediate response to colistin. Colistin has historically been regarded as a last-resort antibiotic, and similar reports of reduced susceptibility in food animals have been documented [1, 49]. This suggests the possible emergence of colistin resistance, which could be driven by the overuse of colistin in animal feeds as a growth promoter and prophylactic agent in Bangladesh [49].

The study further identified that 29.39% of isolates were MDR, with the highest proportion observed in water samples (56.3%), followed by rectal swabs (35.7%). Comparable MDR prevalence has been reported in South Africa with intensive farming systems, often associated with the uncontrolled use of broad-spectrum antimicrobials and cross-contamination within animal environments [13]. Importantly, none of the isolates were XDR or PDR, which suggests that while resistance is a growing concern, there may still be an opportunity to mitigate the spread of extreme resistance through judicious antimicrobial stewardship [50].

This study provides evidence of a high prevalence of class 1 integron (52.57%) among E. coli isolated from food animals in Bangladesh, along with a considerable detection rate of the mcr-1 gene (21.65%). These findings highlight the alarming dissemination of mobile genetic elements and last-resort antibiotic resistance determinants in the animal sector [51]. Our results are consistent with studies from other LMICs, where food animals have been reported as important reservoirs of multidrug-resistant E. coli [52, 53]. For example, study from reported class 1 integron prevalence rates ranging between 45% and 60%, aligning closely with our findings [19]. Similarly, the detection of mcr-1 in 21.65% of isolates is higher than reports from European countries, where prevalence is typically below 10%, but comparable to findings from Southeast Asia, suggesting regional variations linked to antibiotic usage practices in livestock production [52].

A key finding of this study was the significant association of class 1 integron with both MDR and the mcr-1 gene. This aligns with previous reports from Australia and South Africa countries, where class 1 integron has been implicated in the dissemination of resistance genes across diverse bacterial populations [54, 55]. The high prevalence of class 1 integron in MDR isolates suggests that these mobile genetic elements play a critical role in capturing and spreading resistance determinants in food-animal environments [19]. The strong association with the mcr-1 gene is particularly concerning, as integron can facilitate the co-selection of colistin resistance alongside other resistance traits, further limiting treatment options [56].

The detection of mcr-1 predominantly in rectal swabs (25%) and fecal samples (22%) indicates that food animals act as reservoirs for colistin resistance, which may be transmitted to humans through direct contact or via the food chain [27]. The absence of other mcr variants (mcr-2 to mcr-5) in our isolates aligns with studies from South Asia, where mcr-1 remains the predominant variant circulating in animal populations [57].

The co-existence of class 1 integron and the mcr-1 gene in E. coli isolates from cattle highlights the potential for MDR. These findings are consistent with previous studies reporting the presence of mcr-1 in food animals without co-existence with class 1 integron, suggesting that integron may facilitate the accumulation and dissemination of multiple resistance genes [58, 59]. The resistance phenotype was primarily associated with fluoroquinolones and selected β-lactams, underscoring the potential role of integron in mediating MDR even in the absence of phenotypic colistin resistance [17].

Biofilm formation ability, observed in a substantial proportion of isolates, further increases the risk of persistence and resistance dissemination. While our study did not find a statistically significant association between biofilm production and integron carriage, previous studies suggest that biofilm formation can enhance horizontal gene transfer, indirectly promoting the spread of integron and resistance genes [60, 61]. This may partly explain the persistence of MDR E. coli in the environment, particularly in water and fecal samples, as seen in our findings [61].

Limitations of the study

This study has several limitations. First, plasmid carriage and integron context of the isolates were not assessed. Due to financial constraints and the cross-sectional design, we were unable to perform sequencing of integron variable regions, assess plasmids in mcr-1-positive isolates, or confirm gene transfer and genomic perspectives via conjugation assays or whole-genome sequencing. These limitations may affect the comprehensiveness of understanding the genetic mechanisms underlying antimicrobial resistance. We also acknowledge that mapping of integron variable regions, cassette sequencing, and screening for key resistance genes are limitations of our study, and future research should address these aspects to explicitly connect cassette content with per-isolate co-resistance across antibiotic classes. Despite these constraints, our study provides valuable baseline data on resistance patterns in the study setting. A future longitudinal study is needed for a more comprehensive understanding at the national level.

Conclusion

This study demonstrates a high prevalence of class 1 integron and the mcr-1 gene among E. coli isolates from food animals in Bangladesh, highlighting their role in multidrug resistance and the potential spread of colistin resistance. The strong association between class 1 integron, MDR phenotypes, and mcr-1 emphasizes the importance of mobile genetic elements in the dissemination of critical resistance determinants. Additionally, the observed biofilm-forming ability of isolates may further enhance their persistence in animal and environmental sources. Additionally, this study has several limitations. However, these findings underscore the urgent need for prudent antibiotic use in livestock, regular surveillance of resistance genes, and implementation of effective strategies to mitigate the public health risk posed by antimicrobial-resistant bacteria in the food chain.

Abbreviations

AMR

Antimicrobial resistance

AST

Antimicrobial susceptibility testing

ATCC

American Type Culture Collection

BLRI

Bangladesh Livestock Research Institute

CLSI

Clinical and Laboratory Standards Institute

E. coli

Escherichia coli

EMB

Eosin Methylene Blue

ISO

International Organization for Standardization

LB broth

Luria–Bertani broth

MDR

Multidrug resistant

XDR

Extensively drug resistant

PDR

Pan-drug resistant

PBS

Phosphate-buffered saline

PCR

Polymerase chain reaction

intI1

Class 1 integron-integrase gene

mcr

Mobilized colistin resistance gene family

ARRIVE

Animal Research: Reporting of In Vivo Experiments

Authors’ contributions

All authors have thoroughly reviewed and approved the final manuscript, and no qualified individuals have been excluded from authorship. The individual contributions of each author are detailed below: Md. Niamul Shahadat: Conceptualization, Methodology, Microbiological investigation, Data curation, Writing - original draft, Validation. Abu Bakkar Siddique: Conceptualization, Methodology, Microbiological investigation, Data curation, Data analysis and interpretation, Writing - original draft, Validation. Shihab Ahmed: Microbiological investigation, Data curation, Writing - original draft, Validation. Md. Hafizur Rahman: Conceptualization, Methodology, Writing - critical review & editing, supervision, Validation.

Funding

This research was partially funded by small grants from the Bangladesh Livestock Research Institute in Savar, Dhaka, Bangladesh (GOB fund).

Data availability

All data supporting this article are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

In order to respect ethical norms and guarantee the welfare of all participants and biological materials used, this study was conducted in complete accordance with the ARRIVE criteria. The Bangladesh Livestock Research Institute’s (BLRI) Technical Review Committee approved the research protocol (No. AHRD01/2022-23) before to its start, attesting to its compliance with national and international ethical standards. To reduce risk and protect anonymity, every step of the study from sample collection and processing to laboratory analysis was carried out with extreme caution. By encouraging openness, accountability, and respect for moral research methods, the study team stayed dedicated to moral behavior throughout the entire process.

Consent for publication

Not- Applicable.

Competing interests

The authors declare no competing interests.