AbstractThe emergence and spread of multidrug-resistant pathogens, such as Escherichia coli, present major global public health challenges. This study aimed to investigate the prevalence, antibiotic resistance patterns, biofilm production, and the presence of antibiotic resistance genes (ARGs) and biofilm-forming genes in E. coli isolated from fish in open-body water (wild) sources and land-based aquaculture (cultured) systems in Mymensingh, Bangladesh. We collected 130 fish (Koi: Anabas testudineus and Shing: Heteropneustes fossilis) among which 70 were from wild sources and 60 from cultured systems. We screened 116 probable E. coli isolates through selective culture, Gram-staining, and biochemical tests. Using malB gene-specific PCR, we confirmed 87 isolates (67.0%) as E. coli. Cultured fish had a higher prevalence (70.0%) compared to wild fish (64.0%). Biofilm formation was detected in 20.0% E. coli by Congo red agar tests. However, crystal violet assays revealed that 70.0% of E. coli from cultured fish produced biofilm, compared to 20.0% from wild fish, with 7.0% of cultured fish isolates showing strong biofilm production. Antibiotic resistance profiling showed that 100.0% E. coli isolates were resistant to ampicillin and ceftazidime, beta-lactamase-producing antibiotics. Resistance patterns varied by source, with nearly 97.0% of E. coli from cultured fish being multidrug-resistant (MDR), compared to 60.0% in wild fish. E. coli from cultured fish were identified as potential reservoirs of ARGs such as blaTEM (83.0%), blaSHV (81.0%), blaCTX (78.57%), and the biofilm forming gene fimC (100.0%). Significant associations were observed for blaTEM (p = 0.033), blaSHV (p = 0.038), and fimC (p = 0.005). These findings highlight the need for monitoring β-lactamase-resistant and biofilm-forming E. coli in both wild and cultured fish in Bangladesh due to their potential threat to public health and animal populations.
IntroductionEscherichia coliis an exceptionally adaptable microorganism, capable of thriving in a wide range of environmental settings1,2. Its diverse characteristics have made it the most extensively studied microorganism in the world3. E. coliis a natural inhabitant of the gastrointestinal tract in humans and animals4,5,6, where it plays a crucial role in maintaining gut health and digestion. Fecal microorganisms have the potential to endure prolonged periods in soils and manure and water and therefore, they serve as an accessible source of contamination7. It is also found in wastewater, where it can persist under varying conditions, often serving as an indicator of fecal contamination8. Its versatility allows it to survive and multiply in diverse environments, ranging from nutrient-rich to nutrient-poor conditions, making it one of the most studied and significant bacteria in both environmental and medical microbiology1,2.Antimicrobial resistance (AMR) is one of the most significant threats to global health in the twenty-first century9. AMR occurs when microorganisms develop resistance to antibiotics designed to combat them10. This resistance complicates infection treatment due to a lack of effective therapeutics and can lead to multidrug resistance (MDR), posing future challenges in managing infectious diseases11. Overuse of broad-spectrum and unnecessary antibiotics, often without medical guidance, exacerbates resistance issues11,12. The World Health Organization predicts that by 2050, 10 million deaths annually will result from MDR bacteria13. The extensive use of antibiotics in livestock, agriculture, aquaculture, and healthcare sectors promotes the emergence of antibiotic-resistant strains in aquaculture and open-water sources14. Recently, E. colihas shown a growing resistance to many commonly used antibiotics15. E. coliis becoming increasingly difficult to treat due to its evolving resistance to most first-line antimicrobials16. Additionally, resistant E. coli strains can transfer antibiotic resistance genes to other E. colistrains and bacteria within the gastrointestinal tract, leading to the acquisition of resistance from external organisms17. The global prevalence of MDR E. coliin both humans and animals is on the rise2,18,19. The growing resistance of E. colito beta-lactam antibiotics is causing significant challenges in the general population20. Moreover, E. colican develop resistance to various classes of commonly prescribed antibiotics, such as trimethoprim-sulfamethoxazole, aminoglycosides, and fluoroquinolones21. Some E. colistrains produce Extended Spectrum Beta-lactamase (ESBL) enzymes, which can interact with and break down the active compounds in various commonly used antibiotics, rendering them ineffective22,23. Approximately 10% of all E. colistrains isolated worldwide are ESBL-producers22. Another factor contributing to development of MDR in E. coliis its ability to produce biofilms. Biofilms serve as a protective mechanism for bacteria, allowing them to survive in harsh conditions and communicate with one another, forming a collective resistance against antibiotics24. Bacteria adhere to surfaces and secrete Extracellular Polymeric Substances (EPS) to form a protective shell. This biofilm structure makes it difficult for antibiotics to penetrate and effectively kill the bacteria25.Bangladesh, a Southeast Asian country with the highest number of natural water bodies, is home to thousands of fish species26. Fish is a crucial component of Bangladeshi cuisine and culture. In response to the increasing market demand, fish cultivation has expanded through hatcheries and bio-flock systems27. This growth involves many people working in and around the fish industry. Hazardous microorganisms present in these fish pose a potential risk to local populations, particularly those handling, cutting, and cooking fish in the market28. The presence of antibiotic-resistant E. coliin fish not only poses a direct threat to human health through foodborne illnesses but also contributes to the global spread of resistance genes, complicating treatment options for infections29. In aquaculture, the persistence of resistant bacteria, enhanced by biofilm formation, challenges the industry’s sustainability, leading to increased disease management costs and potential economic losses30. Moreover, the discharge of these resistant strains into natural water sources disrupts aquatic ecosystems, further exacerbating environmental degradation30. The presence of MDR E. coli in fish represents a significant global health threat. Despite extensive research on E. coli in commercially farmed fish, there remains a considerable knowledge gap regarding its occurrence in fish from open-body water sources and land-based aquaculture systems in Bangladesh. Furthermore, currently there is a paucity of data concerning MDR E. coli in these environments. This study therefore investigated the prevalence, antibiotic resistance patterns, biofilm production, presence of ARGs, and biofilm forming gene in E. coli isolated from fish populations belonged to open-body water (wild) and land-based aquaculture (cultured) systems in Mymensingh district of Bangladesh, employing both phenotypic and genotypic techniques.ResultsOverall prevalence of E. Coli in wild and cultured fish populationsOut of 130 fish samples, which comprised specimens from both wild sources (n = 70) and cultured systems (n = 60), the overall of prevalence of E. coli was 66.92% (87/130). Specifically, E. coli was detected in approximately 64% (45/70) of the wild fish samples (95% CI: 53–74%) and 70.0% (42/60) of the cultured fish samples (95% CI: 57–80%) (Fig. 1). This analysis reveals a significant prevalence of E. coli in both wild and cultured fish populations. However, bivariate analysis did not detect a significant correlation (p > 0.5) in the prevalence of E. coli between these two types of samples.Fig. 1Prevalence of E. coli isolated from fish in open-body water (wild fish) and land-based (cultured fish) aquaculture systems in Mymensingh, Bangladesh.Full size imageBiofilm formation capabilities of the E. Coli
The Congo Red Agar (CRA) plate test showed low positivity in the presumptive detection of biofilm formation in 20.0% of the analyzed E. coli isolates. Quantitative biofilm formation by E. coli isolates was assessed using a crystal violet assay. Biofilm quantification analyses showed that 80.0% of the E. coli isolates from wild sources did not exhibit any biofilm formation (Fig. 2). Conversely, 70.0% of the E. coli isolates from cultured systems were biofilm producers, indicating that this technique was more efficient than CRA for the detection of biofilm production. Our analysis also revealed that about 20% of the E. coli isolates from both types of samples exhibited intermediate-level biofilm production (Fig. 2). Furthermore, 7% of the isolates from cultured systems were classified as strong biofilm producers, whereas none of the isolates from wild fishes demonstrated strong biofilm production (Fig. 2).Fig. 2Prevalence of biofilm forming E. coli isolated from fish in open-body water (wild fish) and land-based (cultured fish) aquaculture systems. Here, SWF = strong biofilm producer in wild fish, SCF = strong biofilm producer in cultured fish, IWF = intermediate biofilm producer in wild fish, ICF = intermediate biofilm producer in cultured fish, NWF = non-biofilm producer in wild fish, and NCF = non-biofilm producer in cultured fish.Full size imageAntibiotic resistance profile of the E. Coli
The overall results of the Kirby-Bauer disc diffusion method for antibiotic susceptibility testing are illustrated in Fig. 3. Both types of isolates, those from wild source and those from cultured systems, demonstrated significant resistance to various antibiotics (Table S1, and Table S2). Specifically, all isolates from both sample types exhibited 100.0% resistance (95% CI: 95.77–100%) to ampicillin and ceftazidime. Resistance patterns varied for other antibiotics among the isolates from both sources. In isolates from wild fish, approximately 33.0% exhibited resistance to imipenem, while about 66.0% demonstrated intermediate-resistance to ciprofloxacin (Fig. 3a). Bivariate analysis revealed a highly significant positive correlation between ceftriaxone and gentamicin (ρ = 1.000; p = 0.000). Conversely, negative correlations were observed between imipenem and tetracycline (ρ = −0.303; p = 0.043), ceftriaxone and imipenem (ρ = −0.303; p = 0.043), and gentamicin and imipenem (ρ = −0.303; p = 0.043) (Table S3). Additionally, approximately 60.0% of the E. coli isolates from wild sources were classified as MDR, exhibiting resistance to three or more antibiotics from different classes. These isolates showed four distinct MDR patterns, with a multiple antibiotic resistance (MAR) index of 0.2 to 0.4 (Table 1). In addition, approximately 45.0% of these isolates (95% CI: 31.22–60.05%) showed resistance to imipenem (Fig. 3b). Furthermore, around one-third (29.0%) of the isolates from cultured systems demonstrated resistance to both ceftriaxone and fosfomycin. Bivariate analysis revealed a significant positive correlation between ceftriaxone and ciprofloxacin (ρ = 0.615; p = 0.000). Conversely, negative correlations were observed between imipenem and ciprofloxacin (ρ = −0.358; p = 0.020), gentamicin and ceftazidime (ρ = −0.343; p = 0.026), gentamicin and fosfomycin (ρ = −0.325; p = 0.036), and gentamicin and imipenem (ρ = −0.441; p = 0.003) (Table S4). Isolates from fishes belonged to cultured systems exhibited five distinct MDR patterns, with a MAR index ranging from 0.2 to 0.5 (Table 1). Nearly all of these isolates (97.0%) were classified as MDR (95% CI: 87.67–99.87%), as illustrated in Fig. 3c.Fig. 3Antibiotic resistance in E. coli isolates. (a) Antibiotic resistance in E. coli isolates from open body water (wild fish) sources. (b) Antibiotic resistance in E. coli isolates from land-based (cultured fish) aquaculture systems. (c) Multidrug resistance (MDR) patterns in in E. coli isolates sourced from wild fish and cultured fish. Here, TE = Tetracycline, CAZ = Ceftadizime, GEN = Gentamycin, IMP = Imipenem, NIT = Nitrofurantoin, AMP = Ampicillin, C = Chloramphenicol, FF = Fosfomycin, CTX = Ceftriaxone, CIP = Ciprofloxacin.Full size imageTable 1 Multidrug-resistance patterns of E. Coli isolates from open body water (wild fish, no human involvement during raising) sources and land-based aquaculture (cultured fish, cultivated by humans for business purposes in hatchery of ponds) systems.Full size tableAntibiotic resistance and biofilm forming genes in E. Coli
The profiles of antibiotic resistance and biofilm forming genes in E. coli isolates (N = 87) were analyzed to assess the potential risks these strains pose to public health and food safety (Fig. 4a). Using gene-specific polymerase chain reaction (PCR) (Table S5), we analyzed 45 E. coli isolates from fish populations of wild origin for determining the resistance and biofilm production gene profiles (Fig. 4b). PCR results showed that 28 isolates (66.66%, 95% CI: 51.55–78.98%) tested positive for the blaTEM gene, associated with beta-lactamase production. Similarly, 27 isolates (61.36%, 95% CI: 46.66–74.27%) were positive for the blaSHV gene, another beta-lactamase marker (Fig. 4b). The blaCTX gene, linked to extended-spectrum beta-lactamase production, was detected in 28 isolates (66.66%, 95% CI: 51.55–78.98%). Resistance associated with the sul1 gene was found in 20 isolates (47.61%, 95% CI: 33.36–62.27%). Lastly, the biofilm forming gene fimC, associated with fimbrial adhesins, was detected in 40 isolates (95.23%, 95% CI: 84.21–99.15%) (Fig. 4b).Fig. 4Prevalence of antibiotic resistance genes (ARGs; blaTEM, blaSHV, blaCTX and sul1)) and biofilm forming gene (fimC) in E. coli isolated from fish in open-body water (wild fish) and land-based (cultured fish) aquaculture systems. (a) Representative heatmap showing the prevalence of E. coli isolates resistant to different antibiotics and antibiotic-resistant genes. Here, TE = Tetracycline, CAZ = Ceftadizime, GEN = Gentamycin, IMP = Imipenem, NIT = Nitrofurantoin, AMP = Ampicillin, C = Chloramphenicol, FF = Fosfomycin, CTX = Ceftriaxone, and CIP = Ciprofloxacin. (b) Prevalence of ARGs and biofilm forming gene in E. coli isolated from wild fish, and (c) Prevalence of ARGs and biofilm forming gene in E. coli isolated from cultured fish.Full size imageSimilarly, gene-specific PCR (Table S5) was employed to determine the resistance and biofilm production gene profiles in 42 E. coli isolates from fish in cultured systems (Fig. 4c). Our results showed that 35 of these isolates (83.33%, 95% CI: 69.39–91.68%) harbored the blaTEM gene, known for producing beta-lactamase enzyme that helps bacteria resist certain antibiotics. Additionally, 34 isolates (80.95%, 95% CI: 66.69–90.01%) tested positive for the blaSHV gene, another beta-lactamase marker gene. The blaCTX gene was found in 33 isolates (78.57%, 95% CI: 64.04–88.29%). Moreover, 22 isolates of E. coli were found to harbor sul1 (52.38%, 95% CI: 37.72–66.64%) gene. Remarkably, all E. coli isolates (100.0%, 95% CI: 91.62–100.00%) from cultured fish populations were found to harbor the fimC gene (Fig. 4c). This gene is crucial for the production of fimbrial adhesins, which facilitate bacterial adherence to surfaces, thereby indicating a high potential for biofilm formation among these isolates. Furthermore, the bivariate analysis revealed a significant association between the presence of the blaTEM gene (p = 0.033), the blaSHV gene (p = 0.038), and the biofilm forming gene fimC (p = 0.005) in E. coli isolates from fishes belonged to both wild source and cultured aquaculture systems.DiscussionBangladesh, the largest delta of the world, is covered with water for much of the year. Fish play a crucial role in the economy and cuisine of Bangladesh. While previous studies have investigated pathogenic bacteria in fish from Bangladesh31,32,33, there is no published data on the comparative analysis of antibiotic-resistant E. coli and their biofilm formation abilities in fish from closed and open water sources. To address this gap, this study investigated the prevalence, antibiotic resistance profiles, biofilm formation, the presence of ARGs, and virulence genes in E. coli isolates circulating in fish populations sourced from open-body water (wild) and land-based aquaculture (cultured) systems in Mymensingh, Bangladesh, utilizing both phenotypic and genotypic methods. We analyzed 130 fish samples from wild source and cultured systems. Our study revealed that E. coli was present in approximately 67.0% of the tested samples, with a notably higher prevalence in fish from cultured systems compared to those from wild sources. Specifically, E. coliwas detected in 70% of fish collected from cultured systems and 64% of fish from wild sources. In Bangladesh, the majority of fish farms are located in densely populated areas, where farming practices often include the use of poultry droppings as a feed source for farmed fish34. This practice likely contributes to the elevated levels of E. coli detected in samples collected from these farm environments. Supporting this investigation, a study by Ava et al. (2020) reported 75% prevalence of E. coliin fish from the Dinajpur district of Bangladesh, a finding that closely mirrors our results35. This finding suggests that the conditions and practices within these farming setups may play a significant role in the contamination of fish with E. coli. Additionally, a similar prevalence of E. coliwas reported in shrimp samples in Iraq36, indicating that the issue of E. coli contamination in aquaculture may be widespread, influenced by local farming practices and environmental conditions.The quantitative crystal violet microtiter plate (CVMT) tests provided insightful results regarding biofilm formation in E. coli isolates. Specifically, 7% of E. coli isolates from cultured environments were identified as strong biofilm producers, indicating a significant ability to form biofilms in these controlled settings. In contrast, no strong biofilm producers were detected in the isolates from wild sources, suggesting a lower prevalence of robust biofilm formation in these more variable natural environments. These findings contrast with a previous study by Onmaz et al., who investigated reported 24.0% E. coliisolates from fish samples obtained from different markets in Turkey were strong biofilm producers37, highlighting a higher rate of strong biofilm formation compared to our findings. In a recent study from Bangladesh, researchers investigated E. coliin beef and found that 19.0% of their isolates were strong biofilm producers38. While these results indicate a higher prevalence of strong biofilm-producing E. coli compared to our findings, it is important to consider that this difference may be due to variations in sampling locations and sample types. This discrepancy may be attributed to differences in environmental conditions, aquaculture practices, or geographical variations, underscoring the need for localized studies to understand the factors influencing biofilm formation in E. coli across different settings. In addition, it observed variation in the biofilm-forming ability of the isolated E. coli might be under the influence of methodologies used here e.g., Congo Red vs. Crystal Violet assay.The examination of E. coli isolates from fish populations in both wild and cultured sources revealed the presence of various antibiotic-resistant phenotypes. In this study, we evaluated the resistance profiles of E. coliisolates from both wild and cultured fishes against the 10 most commonly used antibiotics in Bangladesh. In this study we detected 66.0% isolates of wild fish as intermediate-resistance to ciprofloxacin. While in a previous study in the same type of fish from Mymensingh, all isolates were found to be sensitive to ciprofloxacin31. This observed variation could be linked to temporal variation. In addition, our findings indicated that all isolates from both types of fish samples were resistant to ampicillin and ceftazidime. While no fosfomycin-resistant E. coliwas found in wild fish, whereas approximately 30.0% of the isolates from cultured fish were resistant to fosfomycin. This profile of AMR may be attributed to the frequent use of antibiotics in fish farming for therapeutic purposes and growth promotion39. The high resistance to ampicillin and ceftazidime is particularly alarming, as these critical beta-lactam antibiotics are commonly used in both livestock, including fish, and human medicine40,41. The detection of antibiotic-resistant E. coliin the studied samples suggests that these fish may serve as spreaders of resistant microorganisms throughout aquatic environments42. Furthermore, 97.0% of isolates from cultured fish exhibited MDR, while approximately 60.0% of isolates from wild fish demonstrated MDR patterns. The widespread and often unregulated use of antibiotics without proper medical prescriptions has led to a concerning increase in antimicrobial resistance among microorganisms in Bangladesh and other Southeast Asian countries in recent years43. A key finding of this study is the investigation of the five most prominent ARGs in E. coli. This information is crucial for developing a surveillance program to help control and reduce the spread of ARGs in the environment. We found that the blaTEM, blaSHV, and blaCTX genes were more frequently present in E. coli isolates from cultured fish, with occurrences ranging from 78.0 to 83.0%. In contrast, these genes were less common in isolates from wild fish populations, where their prevalence ranged from 61.0 to 66.0%. This finding suggests a higher level of antibiotic resistance in cultured fishes, potentially linked to the practices and conditions in cultured aquaculture environments. A previous study conducted by Bora et al. in India reported 78.0% and 89.0% prevalence of the blaTEM and blaCTXgenes44 in E. coli, a finding that aligns closely with our results. This similarity underscores a significant and consistent occurrence of the blaTEM and blaCTX genes across different regions, highlighting its widespread presence in antibiotic-resistant E. coli populations. Another survey conducted by Goudarzi et al. reported a 69% prevalence of the blaTEMgene in their investigation45. This finding is somewhat lower compared to the higher prevalence observed in our study, where blaTEM was found in 78.0–83.0% of the isolates from cultured fish. This discrepancy highlights variations in resistance gene prevalence across different regions and studies, suggesting that local factors may influence the distribution of antibiotic-resistant genes. Such findings emphasize the need for continued monitoring and control measures to address the spread of this resistance gene in both local and broader contexts. Additionally, to identify virulence gene in E. coli, we examined the samples for the biofilm forming gene fimC, which facilitates adhesion of uropathogenic E. coli(UPEC) to epithelial cells during infections46. The biofilm forming gene fimC was present in all (100%) of the fish samples from cultured environments, whereas its prevalence was lower (95.23%) in fish samples from wild sources. Although there is limited public data on the fimC gene in E. coli from fish, a study by Islam et al. reported a 67.0% occurrence of the fimC gene in E. coliisolated from migratory birds in Bangladesh47. It is also important to note that in this study we found inconsistency between the fimC genotype of E. coli and its phenotypic ability to form biofilm. Biofilm formation is a complex mechanism and there are many other genes in addition to fimC involved in the process. Observed inconsistency may be due to involvement of other genes in E. coli biofilm formation.Important limitation of this study is the lack of more samples, however, we designed this study to generating base line data, since no such study was earlier carried out in these fishes in Bangladesh. Koi and Shing are different species of fish. They have different feeding behavior and natural niche adaptation. It is therefore not also unlikely to have variation in load and character of E. coli harbor in these fishes. Many of the variation observed in biofilm formation and AMR in E. coli isolated from wild and cultured environment might also be linked with this variation.ConclusionThis study sought to compare the prevalence, antibiotic resistance, biofilm formation, antibiotic resistance and virulence gene profiles in E. coli isolated from fish populations in wild source and cultured aquaculture systems in Bangladesh, using both phenotypic and genotypic methods. The overall prevalence of E. coli was about 67.0%. The biofilm assay identified 20.0% of E. coli from both sources as positive for biofilm formation. Isolated E. coli showed varying resistance patterns and significant correlations between different antibiotics. MDR E. coli isolates were more prevalent in fish populations from cultured aquaculture systems compared to those from fish populations of wild sources. A significant association was found between blaTEM, blaSHV, and biofilm forming gene fimC. The potential horizontal transfer of these ARGs to other pathogens in both wild sources and cultured aquaculture systems poses serious public health risks. The emergence and spread of MDR E. coli in these environments underscore the urgency for ongoing surveillance and intervention.Materials and methodsSample collection and processingThis cross-sectional study was undertaken at the Bacteriology Laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh. The studied samples were sourced from multiple markets across the Mymensingh district of Bangladesh (Fig. S1). We collected a total of 130 Koi (A. testudineus; size = ± 5 cm, weight = ± 20 g) and Shing (H. fossilis;size = ± 10 cm, weight = ± 18 g) fish. Shing and Koi are the two most abundant types of fish found in local markets and people take them daily as their sources of protein in Bangladesh. Fishermen and consumers frequently encounter these fish and there is a high chance of getting affected by the pathogenic bacteria in those fish. Since these fish are most common and popular in mass people, we selected them as sample for our current study. Among them 70 fish were from open body water sources (40 were Koi fish and 30 were Shing fish) and 60 from culture systems (30 were Koi and 30 were Shing). The samples were immediately transported to the Laboratory, maintaining a cold chain at 4 °C. Upon arrival at the laboratory, the samples were processed under aseptic conditions48. Approximately, 1 g portion from intestine of each fish sample was placed in a mortar and pestle, and ground with Phosphate Buffered Saline (PBS). Subsequently, 1 mL of the homogenized sample was transferred into a 30 mL test tube containing 9 mL of nutrient broth. The tubes were incubated overnight at 37 °C to enrich the target bacteria48,49.Isolation and identification of E. Coli
To isolate E. coli, following overnight enrichment, a loopful (~ 10 µL) of the enriched specimen was aseptically streaked onto Eosin-Methylene Blue (EMB) agar plates (HiMedia, Mumbai, Maharashtra, India) using a sterilized inoculation loop. The plates were then incubated overnight at 37 °C. Colonies displaying a characteristic metallic sheen were presumptively identified as E. coli18,50. These presumptive colonies were subsequently subcultured by transferring them to fresh EMB agar plates to obtain isolated single colonies. Suspected colonies were further checked through Gram staining and a series of biochemical tests, including sugar fermentation, catalase activity, indole production, and the Voges–Proskauer test2,51. Thereafter, pure E. coli colonies of 116 presumptive isolates (wild fish = 64 and cultured fish = 52) were preserved in 20% glycerol and stored at – 20 °C for future use.Molecular detection of E. Coli
The isolates were molecularly confirmed as E. coli using a species-specific PCR (Table S5) method targeting the malB gene, which encodes the maltose-binding protein. This gene is specific to E. coliand serves as a reliable marker for its identification52. PCR amplification was carried out using malBF (5´-GACCTCGGTTTAGTTCACAGA-3´) and malBR (5´- CACACGCTGACGCTGACCA-3´) primers2,50, and the presence of a PCR product of the expected size confirmed the identity of the isolates as E. coli. Genomic DNA was extracted from an overnight culture using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The quality and quantity of the extracted DNA were assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). In each PCR around 50 ng DNA was used as template, while the primer concentration was 20 pmol for each. DNA samples with A260/280 and A260/230 ratios of approximately 1.80 and 2.00 to 2.20, respectively, were considered high-purity53and stored at – 20 °C prior to PCR amplification50,51. The PCR amplification of the targeted DNA was conducted in a 20 µL reaction mixture in a gradient thermal cycler (Blue-Ray Biotech Corp., Taiwan). This mixture consisted of 3 µL of nuclease-free water, 10 µL of 2X master mix (Promega, Madison, WI, USA), 1 µL each of forward and reverse primers, and 5 µL of the DNA template. To ensure the accuracy of the PCR results, positive controls consisting of E. coligenomic DNA, which had been previously confirmed for the target genes, were included in the reaction50. PCR-negative controls used non-template controls, where PBS was included instead of genomic DNA. The amplified PCR products were subjected to electrophoresis on a 1.5% agarose gel and visualized using an ultraviolet transilluminator (Biometra, Göttingen, Germany). A 100 bp DNA ladder (Promega, Madison, WI, USA) was utilized to verify the expected sizes of the PCR products49,50. Finally, 87 isolates, comprising 45 from wild fish and 42 from cultured fish, were identified as E. coli through species-specific PCR.Biofilm formation in E. Coli
Qualitative methodThe qualitative assessment of biofilm-producing E. coli was conducted using the CRA plate method. In this method, E. coli isolates (N = 87) were cultured on CRA plates, which contain Congo Red dye. The biofilm production capability of the isolates was evaluated based on the phenotypic characteristics of the colonies (Fig. S2)54. To prepare CRA plates, 56 g of Brain Heart Infusion (BHI) agar was dissolved in 1 L of double-distilled water. After adding 0.8 g of Congo Red dye and 36 g of sucrose, the solution was gently heated until all components were fully dissolved. The mixture was then sterilized by autoclaving at 121 °C and 15 per square inch (psi) for 30 min51. Once autoclaved, the media was poured into sterilized Petri dishes, allowed to cool to room temperature, and incubated to ensure sterility. For assessing biofilm formation, a loopful of bacterial suspension was streaked onto the CRA plates and incubated overnight at 37 °C, followed by an additional 24 h at room temperature. Colonies that appeared as robust, crusty, black were classified as strong biofilm producers, while red colonies were identified as non-biofilm-producing strains. All the experiments were repeated twice.Quantitative methodFor the quantitative assessment of biofilm production by E. coli (N = 87 isolates), the CVMT method, as described by Rana et al.48, was employed. A single colony from a CRA plate was inoculated into a 1.5 mL Eppendorf tube containing nutrient broth supplemented with 2% sugar and incubated overnight at 37 °C. Subsequently, the biofilm formation assay was performed using a 96-well microtiter plate. Each well was filled with 180 µL of Tryptic Soy Broth (TSB) supplemented with 2% sugar, and 20 µL of the adjusted overnight culture (McFarland 0.5 standard) was added51. The microtiter plate was incubated at 37 °C for 24 h. A control well containing 200 µL of TSB without test samples was included for comparison. After incubation, the wells were thoroughly washed with distilled water to remove non-adherent planktonic bacteria. The biofilms were then stained with 1% crystal violet, and the plates were allowed to air dry. Biofilm quantification was achieved by measuring the absorbance at 570 nm using an ELISA reader24. The data were interpreted according to established criteria for biofilm formation.Antibiotic susceptibility assayThe Antibiotic Sensitivity Test (AST) of the PCR-positive E. coli isolates (N= 87) were assessed using the Kirby-Bauer disk diffusion method (DDM)55, in accordance with the guidelines outlined in the Clinical Laboratory Standards Institute (CLSI) M100 33rd Edition56. We selected the ten most frequently used antibiotics in Bangladesh from different groups.These were ciprofloxacin (CIP, 5 µg), gentamicin (GEN, 10 µg), tetracycline (TET, 30 µg), ceftriaxone (CTR, 30 µg), ampicillin (AMP, 25 µg), ceftazidime (CAZ, 5 µg), chloramphenicol (C, 30 µg), imipenem (IMP, 10 µg), fosfomycin (FOS, 50 µg), and nitrofurantoin (NIT, 300 µg). The isolated colonies were taken into 4–5 mL of nutrient broth for performing DDM. After preparing the broth cultures, they were incubated for 4–5 h at 37 °C, and the turbidity of bacterial suspensions was adjusted with the 0.5 McFarland unit (HiMedia, India). After that, the dried surface of a Muller Hilton (MH) agar plate was inoculated by spreading the broth suspension on the surface with sterile cotton swabs. Finally, the antibiotic disks were applied on the surface of the agar plates and left for overnight incubation at 37 °C. The isolates were categorized as susceptible, intermediate, and resistant according to CLSI guidelines56. MDR patterns, defined as resistance to > 3 antibiotics, were identified using the protocol i.e., any isolate not susceptible to at least one agent in at least three antimicrobial classes outlined by Sweeney et al.57. The Multiple Antibiotic Resistance (MAR) index was calculated by the following formula: MAR = u/v; where, u = total number of antibiotics that an isolate showed resistance and v = total number of antibiotics used in this study58. E. coli strain ATCC25922 was used as the negative control in the antimicrobial susceptibility tests.Molecular detection of antibiotic resistance and biofilm forming genes in E. Coli
To identify antibiotic resistance and virulence genes in the E. coli isolates (N = 87), we performed conventional PCR assays targeting beta-lactam resistance genes (e.g., blaTEM, blaSHV, blaCTX, and sul1) and the biofilm forming gene fimC using specific primers (Table S5). Genomic DNA from E. coli isolates was extracted from overnight cultures using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). The quality and quantity of extracted DNA were assessed as previously detailed in Sect. 2.350,51. PCR was carried out using a gradient thermal cycler (Blue-Ray Biotech Corp., Taiwan). Each PCR reaction consisted of a 10 µL mixture, including 5 µL of 2X master mix (Promega, Madison, WI, USA), 2 µL of genomic DNA (50 ng/µL), 0.5 µL of each primer, and 2 µL of nuclease-free water. The PCR products were then analyzed by electrophoresis on a 1% agarose gel. After amplification, the amplicons were stained with ethidium bromide and visualized under an ultraviolet transilluminator (Biometra, Göttingen, Germany). A 100 bp DNA ladder (Promega, Madison, WI, USA) was used to verify the size of the PCR amplicons49,50. Although, a positive control was not used in the PCR for resistance and virulence genes, non-template control (NTC, no template DNA) was used as a negative control.Statistical analysisData were entered into Microsoft Excel 2020 (Microsoft Corp., Redmond, WA, USA) and analyzed using SPSS version 25 (IBM Corp., Armonk, NY, USA), Origin 2024b, GraphPad Prism version 8.4.3 (GraphPad Software, Inc.) and R packages. The Pearson’s chi-square test was conducted to compare the occurrence of E. coliacross different sample categories (e.g., koi fish and shing fish). Prevalence percentages were calculated by dividing the number of positive samples in each category by the total number of samples tested within that category59,60. The prevalence formula was applied for determining occurrence percentage of E. coli. The AMR patterns, resistance, intermediate and sensitivity, and MAR index were calculated using the CLSI (2023) guideline using the cut-off as provided in the brochure of the manufacturer (Liofilchem, Italy).
Data availability
All data generated or analyzed during this study are included in this article [and its supplementary information files].
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Download referencesAcknowledgementsThe authors would like to thank the Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202 for the support during the present research.FundingThis research was partially funded by the Bangladesh Agricultural University Research System (BAURES) (grant No. 2022/12/BAU).Author informationAuthors and AffiliationsDepartment of Microbiology and Hygiene, Faculty of Veterinary Sciences, Agricultural University, 2202, Mymensingh, BangladeshMd. Liton Rana, Md. Ashek Ullah, Jayedul Hassan, Mahbubul Pratik Siddique & Md. Tanvir RahmanMolecular Biology and Bioinformatics Laboratory, Department of Gynecology, Obstetrics and Reproductive Health, Faculty of Veterinary Medicine and Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural University, 1706, Gazipur, BangladeshM. Nazmul HoqueNational Engineering Research Center of Industrial Wastewater Detoxication and Resource Recovery, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, 100085, Beijing, ChinaMd. Liton RanaUniversity of Chinese Academy of Sciences, 100049, Beijing, ChinaMd. Liton RanaAuthorsMd. Liton RanaView author publicationsYou can also search for this author in
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PubMed Google ScholarContributionsM.L.R., M.N.H. and M.T.R. conceived and designed the study; M.L.R., and M.A.U. acquisition of data, analysis and writing original draft; M.N.H., J.H., M.P.S. and M.T.R. critical review and editing. All authors contributed to the article and approved the submitted version.Corresponding authorCorrespondence to
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Ethical approval
The animal study protocol was approved by the Ethics Committee of Bangladesh Agricultural University (BAU) in Mymensingh, Bangladesh [AWEEC/BAU/2023(25)]. All experimental procedures and methods were conducted in strict compliance with relevant guidelines and regulations, and the findings were documented in accordance with the ARRIVE guidelines (https://arriveguidelines.org). Notably, this study did not involve the use of anesthesia or euthanasia methods.
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Reprints and permissionsAbout this articleCite this articleRana, M.L., Ullah, M.A., Hoque, M.N. et al. Preliminary survey of biofilm forming, antibiotic resistant Escherichia coli in fishes from land based aquaculture systems and open water bodies in Bangladesh.
Sci Rep 15, 7811 (2025). https://doi.org/10.1038/s41598-024-80536-6Download citationReceived: 15 August 2024Accepted: 19 November 2024Published: 06 March 2025DOI: https://doi.org/10.1038/s41598-024-80536-6Share this articleAnyone you share the following link with will be able to read this content:Get shareable linkSorry, a shareable link is not currently available for this article.Copy to clipboard
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Keywords
E.coli
Biofilm productionAntibiotic resistanceWild and cultured fishPublic healthBangladesh