Distribution and antibiotic resistance patterns of Pseudomonas aeruginosa across different point sources of pollution in the Buriganga River, Bangladesh

The Buriganga River, a vital water body in the capital of Dhaka city in Bangladesh, serves as a critical resource for various activities, including drinking water supply, transportation, cleaning, washing, recreation, ground water recharge, flood control, agriculture, and industrial processes. However, due to rapid urbanization and industrialization in the region, and weaknesses in government law enforcement, wastewater from various industries located beside the river such as battery manufacturing, garment factories, steel mills, and tanneries is directly discharged into the river, which has contributed to the deterioration of water quality, leading to its designation as the biologically and hydrologically ‘dead’ river in Bangladesh. The pollution in the Buriganga River carries significant health implications for the surrounding communities, primarily due to the presence of toxic substances and pathogens in the water (Kormoker et al. 2023). Various points have been identified as sources of pollution (point source) along the river such as discharge from industrial effluent and discharge from municipal and waste water. Many industrial facilities near the Buriganga River lack their own sewage treatment plants. Each day, over 60,000 cubic meters of hazardous waste, including runoff from textile dyeing, printing, washing, and pharmaceuticals, is discharged into the primary water bodies of Dhaka (Rahman & Rana 1995; Alam & Marinova 2003; Jolly et al. 2023). Additionally, many other diffuse sources of pollution such as diffuse farming pollution, urban and transport runoff, dumping of solid wastes along the banks by river side dwellers, leakage of oil from floating oil-seller boats and direct disposal of wastes (like residue of food, human excreta) have been identified as sources of pollution (Kibria et al. 2015). Due to the inadequacy of Dhaka city's sewerage network, there has been a tendency for unauthorized domestic sewer connections to be made into the stormwater pipes, which drain into the Buriganga River. This causes discharge of massive amounts of untreated municipal wastewater and sewage directly into the Buriganga through the city drains and sewers. Several studies report that approximately 900 cubic meters of untreated domestic and human waste are discharged into the Buriganga-Turag system each day (Rahman & Rana 1995). According to Rahaman and Rana the major sources of pollution in the Buriganga River are several industrial installations, municipal wastewater and sewage treatment plants.

Recent research on river contamination and point-source pollution have several key areas, driven by growing environmental concerns, one of which is microbial contamination and antibiotic resistance (Larsson & Flach 2022). Rivers are considered reservoirs of antibiotic-resistant bacteria (ARB), where pollution with other contaminants, such as heavy metals and residual antibiotics, can lead to the dissemination of ARB in the rivers. The Buriganga River has accumulated a diverse array of contaminants, with notable quantities of antibiotics and antimicrobial agents present (Parvin et al. 2022). Improper disposal of unused medications from households and healthcare facilities has also been identified as an additional source of antibiotics in the river (Hossain et al. 2018). In Dhaka City, heavy metals are often discharged from various anthropogenic sources, including textiles and tanneries, leading to the contamination of the Buriganga River. This contamination results in the accumulation of metals such as chromium (Cr), cadmium (Cd), lead (Pb), nickel (Ni), and zinc (Zn) in surface waters (Akbor et al. 2020; Majed et al. 2022). A recent report by Kormokar et al. 2023, showed that the river water is unsafe for residential and recreational use, and the high concentration of heavy metals in the river could affect the ecological balance in the future (Kormoker et al. 2023). Moreover, the survival mechanisms of bacteria under these conditions, in the presence of heavy metals, may also provide selective pressure for ARB.

One of the most alarming concerns regarding rivers in relation to ARBs is the spread of ARBs through the environment and, subsequently, into human populations. A water quality assessment of the Buriganga River reported it to be highly contaminated with toxic chemicals and microbial entities, making it unfit for human consumption. A study by Ghosh et al. (2023) suggests that inhabitants living near the Buriganga River are at serious health risk, including respiratory infections, liver cirrhosis, renal impairment, tissue necrosis, and other toxicities. The presence of Pseudomonas aeruginosa, an opportunistic pathogen commonly found in environments contaminated with organic and inorganic pollutants, particularly in water bodies like the Buriganga River, further exacerbates these health risks (Ghosh et al. 2023). While studies on the prevalence of antibiotic-resistant P. aeruginosa in the Buriganga River have not yet been conducted, similar urban rivers often show significant levels of this bacterium, particularly in areas with high pollution loads, hydrocarbons, and poor sanitation practices (Crone et al. 2020). The presence of P. aeruginosa in water bodies is concerning, as it is known for its antibiotic resistance and potential to cause infections, especially in immunocompromised individuals.

A large population residing near the Buriganga River relies on its water for daily needs, potentially exposing themselves to P. aeruginosa infections. This bacterium can be transmitted through diluted antiseptics, washing liquids, soaps, cosmetics, and even life-support equipment, thereby facilitating person-to-person transmission. While the primary mode of acquisition for P. aeruginosa is environmental, person-to-person spread is rare. However, the prevalence of antibiotic-resistant strains of P. aeruginosa in river water poses a significant threat to both public health and environmental sustainability in Bangladesh. Controlling the dissemination of antibiotic-resistant P. aeruginosa in river water requires a multifaceted approach that addresses the sources of contamination and promotes sustainable practices. Therefore, understanding the factors driving the prevalence of antibiotic-resistant P. aeruginosa in the Buriganga River is crucial for developing effective strategies to combat the spread of antibiotic resistance and safeguard public health.

The present study was designed to investigate the distribution and prevalence of antibiotic resistance patterns of P. aeruginosa in river water among seven-point sources of contamination in the River Buriganga, Bangladesh.

Surface water samples were collected from March 1 to May 15, 2023, during the pre-monsoon season, at seven designated points along the Buriganga River, labeled as A, B, C, D, E, F, and G. These sampling sites were selected based on their proximity to various point sources of pollution. The point sources represent areas where different types of pollution enter the river, including plastic waste, oil spills, domestic waste, sewage, medical waste, and chemical runoff from factories and boat dockyards.

After collecting, water samples were transported to the laboratory, and pH of the water samples were measured and processed within 2 h. Initially, 100 mL of water sample from each sampling point was filtered using a filter paper (Whatman) to remove visible pollutants. To retain bacteria from river water, the filtered water was subsequently vacuum filtered through a 0.45-μm nitrocellulose membrane (Nitrocellulose membrane filter, Sartorius, Germany). A selective medium, Cetrimide agar (Oxoid, UK), was used for isolation of P. aeruginosa from river water without any prior enrichment stage. This was done to reflect the natural state of the bacterial population in the ecosystem. The nitrocellulose filters were then placed onto the cetrimide agar plates, and the plates were incubated at 35–37 °C for up to 48 h after which plates observed for the distinctive colony characteristics of P. aeruginosa. Colonies of P. aeruginosa on cetrimide agar typically appear as medium-sized, irregular-shaped colonies with a characteristic yellow-green to blue coloration, resulting from the production of two primary pigments: pyocyanin (greenish-blue) and fluorescein (yellow-green). For further evaluation and detection, the plates were observed under a UV illuminator for colonies exhibiting a distinctive blue-green fluorescence, a characteristic feature of P. aeruginosa. However, it should be noted that several other fluorescent species of Pseudomonas, such as  Pseudomonas putida or  Pseudomonas fluorescens, may exhibit similar fluorescence under UV light. While both of these other fluorescent species produce yellow-green fluorescence, they do not produce the characteristic blue-green fluorescence due to the pigment pyocyanin, which is a key distinguishing factor for P. aeruginosa. After identification colonies were selected and streaked on cetrimide agar. Pure cultures selected colonies of P. aeruginosa were subsequently prepared and stored on non-selective nutrient agar (Nutrient Agar, HiMedia, India) for further study.

The standard agar disk diffusion method known as the Kirby Bauer method (Hudzicki 2009) was used to determine the antibiotic susceptibility profiles of isolated P. aeruginosa according to the recommendations of the Clinical and Laboratory Standard Institute. The potencies of the antibiotics used against P. aeruginosa are listed in Table 1.

A 24-h-old pure culture of P. aeruginosa isolates was used to inoculate 5 mL of fresh broth at 37 °C for 4–5 h, until it reached the turbidity of a 0.5 McFarland standard. The culture was then used to create a suspension in normal saline, standardized to a 0.5 McFarland turbidity. Sterile cotton swabs were dipped into the suspension, and excess fluid was removed by rotating the swabs firmly against the inside of the tube, just above the liquid level. The dried MH agar plate was then streaked six times with the swabs, rotating the plate 30 degrees after each streak to ensure even distribution of the inoculum. Any excess liquid was collected by rimming the plate with the swabs before discarding them. Antibiotic disks were placed over the inoculated media surface with sterile forceps. The plates were incubated at 37 °C for 18–24 h. After incubation, the plates were examined, and the inhibition zone diameters were measured in millimeters using a ruler. The zone diameters were interpreted as susceptible (S), intermediate resistant (I), or resistant (R) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (details in Supplementary material). Isolates that were resistant to at least one antimicrobial agent in three or more different classes of antibiotics were considered multidrug-resistant (MDR). The reference strain P. aeruginosa (ATCC 27853) was used for quality control.

Forty-eight isolates (96%, 48/50) were found to be resistant to at least one antibiotic assessed. Resistance to three or more antimicrobials was observed in 26% (13/50) of P. aeruginosa isolates (details in Supplementary material). These isolates were all classified as MDR. P. aeruginosa isolates exhibited five multidrug resistance patterns, ranging from three to six antimicrobial agents in three to five antimicrobial categories (Table 4). Most of the MDR isolates were resistant to four antimicrobial agents (61.54%, 8/13). Three isolates (23.08%, 3/13) showed resistance to three antibiotics. One isolate (7.69%, 1/13) was resistant to five antimicrobial agents, and one isolate (7.69%, 1/13) was resistant to six antimicrobial agents. The most common resistance pattern was cefotaxime–cefepime–tetracycline–ciprofloxacin, observed in eight isolates (61.54%, 8/13). No extensive drug-resistant (XDR, resistant to at least one antimicrobial agent in all but two or fewer antimicrobial categories) or pandrug-resistant (PDR, resistant to all antimicrobial agents in all categories) strains were found in this study.

P. aeruginosa is a common bacterial pathogen found in a variety of environmental samples. Despite breakthroughs in medical and surgical care, as well as the use of a wide range of antimicrobial drugs in animals and agriculture, the number of resistant isolates in environmental surface water is increasing dramatically. As a result, various water sources may develop a widespread pool of resistant microorganisms from which resistance may be transferred back into humans and animals. As a result, the current investigation was conducted to determine the proportion of resistant isolates in surface water and to screen their antibiotic resistance pattern.

In our study, colonies of P. aeruginosa on cetrimide agar appeared as medium-sized, irregular-shaped colonies with a characteristic blue coloration, indicative of pyocyanin production (greenish-blue). For further evaluation, the plates were examined under a UV illuminator, where colonies exhibiting distinctive blue-green fluorescence were observed, a key diagnostic feature of P. aeruginosa. This method is advantageous due to its simplicity, speed, and ability to distinguish P. aeruginosa from non-fluorescent or less fluorescent species in mixed microbial communities. However, while the presence of fluorescence provides an initial indication, other fluorescent species, such as P. putida and P. fluorescens, also produce fluorescence under UV light, which could lead to misidentification. Therefore, confirmatory tests are essential to differentiate P. aeruginosa from other fluorescent species. Future studies should incorporate additional confirmatory methods, such as biochemical tests or molecular techniques, to validate the presence of P. aeruginosa and further investigate its potential for antibiotic resistance.

Antibiotic resistance was prevalent at all sampling sites; however, there were variations among different sampling points. These variations could reflect differences in water quality due to the presence of various waste contaminants (Liu et al. 2023). Our findings also indicate that most of the MDR isolates were obtained from upstream sites, where three main point sources of waste – hospitals, tanneries, and textile industries – along with municipal waste, which are known to enter the river, contribute to the contamination (Uddin & Jeong 2021). The area surrounding these sources could be a hotspot for the dissemination of MDR bacteria downstream of the river.

Our study shows the highest percentage of MDR P. aeruginosa isolates (41.67%) from site F. This site has a nearby source of untreated waste from tanneries and textile industries. Although the exact amount of tannery and textile waste discharged into the river at this site is unknown, reports indicate that the tannery sector produces approximately 20,000 m³ of liquid waste and 232 tons of solid waste per day (Alamgir et al. 2017). Furthermore, according to a report from the Department of Environment (DoE), the textile industry was estimated to produce 349 million cubic meters of wastewater in 2021 (Sakamoto et al. 2019). Some components in these wastes, such as heavy metals, dyes, organic solvents, and surfactants, can contribute to the dissemination of MDR in bacteria. Heavy metals such as chromium, lead, cadmium, and mercury and also organic solvents, hydrocarbons and nitrogen- and phosphorus-rich compounds in these wastes are found to be discharged in to the river (Alam et al. 2020). P. aeruginosa, being versatile bacteria, are well known to survive in the presence of these contaminants and some are also capable of tolerating heavy metals like lead (Vélez et al. 2021; Alfarras et al. 2022). A separate study conducted by Ramos et al. showed 91% of MDR isolates of Pseudomonas isolates from lakes, streams and rivers in Brazil showed tolerance to heavy metals and Chromium (Cr) (Ramos et al. 2020), a highly toxic chemical element, mainly introduced in to the river from tanning and dyeing industries (Rahman & P 2019). Chromium can contribute to the co-selection of antibiotic resistance on bacterial populations, by affecting their electrochemical systems and promoting the horizontal transfer of antibiotic resistance genes (Mahmud et al. 2024). A study conducted in Dhaka city showed a high prevalence of P. aeruginosa in industrial and tannery wastewater samples and found that isolates were resistant to most commercial antibiotics, confirming the MDR of P. aeruginosa in wastewater.(Tarannum et al. 2024). These findings support the possible relation of increased prevalence of MDR with the type of waste recovered from site F in the River Buriganga highlighting the importance of proper waste management system in this area.

A separate study identified MDR P. aeruginosa isolates in medical waste, demonstrating a particularly high level of resistance to several commonly used antibiotics (Hasan et al. 2020). This study supports our finding of MDR P. aeruginosa near the point source of medical waste at site E, where untreated medical waste is dumped nearby and could be introduced into the river. Adjacent to site E is Sir Salimullah Medical College (SSMC) Hospital, established in 1960. It is one of the oldest medical colleges in the country and provides medical education and training to students. Improper management of medical waste from this site, discharged into the river, can have severe impacts on both human health and the environment. Antibiotics from these medical sources, as well as from sewage (e.g., point source D in our study), can contaminate the river. Although no current reports are available on the types of residual antibiotics at this location, we hypothesize that various residual antibiotics may be present due to mismanagement of waste disposal systems and the unregulated use of antibiotics in the country. The high prevalence of MDR P. aeruginosa in this area may be attributed to the selective pressure exerted by the antibiotics, promoting the proliferation of multidrug-resistant strains. These resistant P. aeruginosa strains, capable of thriving in contaminated environments, can also spread to human populations, further exacerbating public health risks. Concentrations of residual antibiotics in river water can disrupt natural microbial communities, selecting for resistant strains over time (Maruzani et al. 2020).

In our study, although antibiotic resistance was prevalent in other sources such as plastic waste, household waste, and ship dockyards along the river, the isolates from these sources could not be classified as MDR. However, recent research on microplastics suggests that they can serve as vectors for bacteria, potentially harboring and disseminating antibiotic-resistant strains through environmental exposure (Du et al. 2024).

This study reflects the prevalence of MDR bacteria during the summer season. However, continuous monitoring is essential to track changes in MDR levels over time and identify contributing factors that may influence their prevalence at various point sources. The Buriganga River exhibits distinct seasonal variations: during the monsoon (rainy season), soluble and insoluble waste materials are washed into the river from nearby khals, canals, and surrounding areas. In contrast, during the dry season (winter), the river water becomes stagnant, and the direct discharge of untreated waste from industries, households, and medical facilities significantly worsens pollution levels. Given these seasonal changes, it is possible that the prevalence of MDR bacteria could be higher during the winter months, when water quality deteriorates further due to stagnation and increased contamination.

In the present experiment, piperacillin-tazobactam and gentamicin were the most effective antibiotics, exhibiting 100 and 98% sensitivity, respectively. Imipenem, meropenem, and amikacin followed with sensitivities of 96, 94, and 92%, respectively. Ciprofloxacin and cefepime demonstrated moderate susceptibility, with 62 and 46% of the isolates being sensitive, respectively. Cefotaxime and tetracycline were the least effective, with only 4% sensitivity. These results align with recent reports highlighting rising antimicrobial resistance in environmental isolates, particularly for ciprofloxacin, which saw an increase of 24% in resistance (Nasreen et al. 2015). Notably, 88% of isolates in the present study were resistant to both cefotaxime and tetracycline, mirroring findings from other global rivers (Singh et al. 2019).

Of particular concern, 4% of the isolates in this study were resistant to both meropenem and imipenem, marking a deviation from previous studies in Bangladesh, which reported 100% susceptibility to these carbapenems (Bhuiya et al. 2018). In recent studies, single carbapenem resistant isolate of P. aeruginosa was recovered from urban zone samples of river water in La Rioja region, Spain (Rojo-Bezares et al. 2024). Similarly, two carbapenemase (SPM-1)-producing healthcare-associated critical-priority P. aeruginosa were isolated from impacted urban rivers in São Paulo, Brazil (Esposito et al. 2021). Both sources having possible association with hospital effluents discharged into the river. This highlights the emerging threat of carbapenem-resistant strains in environmental sources such as river water. Additionally, 2% of isolates were resistant to amikacin, which is lower than the 11.5% resistance rate reported in hospital wastewater studies (Roulová et al. 2022). These findings underscore the growing concerns about the spread of antimicrobial-resistant bacteria in environmental settings, particularly in water sources subjected to contamination from human activities.

P. aeruginosa isolates from environmental samples are increasingly developing resistance to antibiotics. This may be attributed to the bacterium's inherent resistance to certain antibiotics, as well as its ability to acquire resistance genes through various mechanisms such as mutation, horizontal gene transfer, and epigenetic changes. The prevalence of MDR isolates has notably increased in recent years. For instance, a study conducted in India in 2012 identified 7.2% MDR P. aeruginosa isolates in both clinical and environmental samples (Sivaraj et al. 2012). In contrast, a study in Japan in 2013 found no MDR P. aeruginosa isolates among hundreds of isolates from two rivers (Suzuki et al. 2013). On the other hand, studies focusing on clinical isolates in Nepal and Malaysia revealed much higher proportions of MDR isolates, with 20.69 and 19.6%, respectively (Pathmanathan et al. 2009; Anil & Shahid 2013). These findings underscore the variation in MDR prevalence across geographic regions and between clinical and environmental sources. In the present study, 26% of the isolates (13 out of 50) exhibited multidrug resistance. These findings underscore the urgent need for effective control measures to limit the spread of MDR P. aeruginosa through the riverine system, particularly through improved waste management practices.

The relatively high resistance of P. aeruginosa isolates to commonly used antibiotics, as observed in this study, is concerning, particularly in developing countries like Bangladesh, where many of these antibiotics are still used as first-line treatments. Our findings indicate that P. aeruginosa is becoming progressively less responsive to cephalosporins, fluoroquinolones, and tetracyclines. Furthermore, there is a significant risk of the spread of carbapenem-resistant P. aeruginosa in the river, where it may be transmitted to other environmental sources through multiple mechanisms. The unregulated use of antibiotics and the lack of proper disposal systems for medical and industrial waste in Bangladesh are urgent issues that need to be addressed. Comprehensive epidemiological surveillance of P. aeruginosa is crucial to prevent the transmission of antibiotic resistance to humans and animals via water systems.

The present study found that antibiotic-resistant P. aeruginosa is prevalent in the Buriganga River water. A high prevalence of MDR strains was observed near the following three key point sources: chemical waste, medical waste, and sewage disposal points. This suggests that these sources may be significant contributors to the dissemination of MDR P. aeruginosa in the river. Addressing point-source contamination, particularly in relation to antibiotic-resistant P. aeruginosa, requires targeted strategies that focus on specific locations or activities contributing to the pollution. The study underscores the importance of strengthening regulations and enforcing stricter laws regarding the discharge of wastewater from industries, hospitals, and sewage treatment facilities. Such measures are crucial to limit the release of antibiotics and resistant bacteria into the river, which could otherwise exacerbate public health risks.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.