Bacteriological assessment of water quality in Barishal, Bangladesh: potability testing and antibiogram profiling

Water is essential for all living entities, crucial for the formation and rejuvenation of cells, and irreplaceable in the sustenance of life on our planet (Ball 2017). Accordingly, a consistent and abundant provision of water is imperative for the survival of biological entities. Surface water bodies remain the main source of drinking water, accounting for nearly half of global demand (WHO 2016). Access to clean and safe drinking water is essential for human health and well-being. Regrettably, numerous regions throughout the globe encounter difficulties in maintaining the quality of their water supplies (Ashbolt 2004; Mabvouna et al. 2020).

According to the World Health Organization (WHO), approximately 827,000 fatalities are ascribed to the precariousness of water, inadequate sanitation, and insufficient hygiene practices each year in low- and middle-income countries (WHO 2019). Water contamination attributed to various types of microorganisms is the leading factor in 80% of human diseases in developing countries (WHO 1993). Water sources face various contamination hazards, including the presence of animal feces, the practice of open defecation, and pollution from on-site sanitation facilities like pit latrines and septic systems, as well as urban runoff (Chigor et al. 2012; Okullo et al. 2017). Water contamination caused by fecal matter from domestic and human excreta presents a notable public health hazard that can transmit different waterborne diseases through the fecal-oral route (Ashbolt 2004; Kostyla et al. 2015).

Recent studies have increasingly drawn attention to the rising prevalence of antibiotic-resistant bacteria (ARB) in water sources, further exacerbating the global public health challenge (Hendriksen et al. 2019). Contaminated water, containing microbiota present in the fecal contents of both animals and humans, may facilitate the horizontal transfer of antibiotic-resistant genes. Subsequently, the ingestion of contaminated water by both humans and animals serves as intrinsic habitats and reservoirs for ARB and their associated resistance genes, contributing to the further dissemination of antibiotic resistance (Okeke et al. 2005; Jiang et al. 2022). The environmental spread of ARB through water systems poses a dual threat: not only does it increase the risk of waterborne infections, but it also promotes the propagation of antimicrobial resistance, complicating treatment strategies for bacterial infections.

There are different sources of drinking water in Bangladesh, such as groundwater, surface water, pipe water, rainwater, and others. However, the supply of clean and safe drinking water still remains a significant challenge in Bangladesh, particularly in rural and semi-urban areas. Therefore, over 97% of the rural population relies on groundwater for their water supply. The insufficient availability of surface water, coupled with pollution, industrialization, and urbanization, has fostered a widespread perception of the safety of groundwater. This has led the majority of people, both in urban and rural areas, to prefer it for their water consumption (Hasan et al. 2019). Unfortunately, both surface and groundwater exhibit significant contamination with diverse pathogens, leading to a range of waterborne diseases such as hepatitis, intestinal infections, diarrhea, dysentery, cholera, typhoid, jaundice, and skin diseases (Kabir et al. 2019; Parvin et al. 2022). Moreover, the presence of ARB in drinking water sources has been linked to the misuse of antibiotics in human health, animal farming, and aquaculture, further intensifying the risk of AMR transmission within communities.

Numerous studies have evaluated the water quality and safety standards across various regions in Bangladesh (Islam et al. 2010; Acharjee et al. 2014; Datta et al. 2014), but there has been no assessment of the quality and safety standards of different water bodies in and around Barishal city. Significant gaps remain in the research on the bacteriological quality and safety of water sources commonly used by the public in this area. Additionally, limited data exist regarding the prevalence and diversity of ARB in Barishal's water sources, making it crucial to investigate their presence and resistance patterns. To fill these gaps, this study focused on the bacteriological quality of water, including potability testing to determine if the water is safe to drink and antibiotic resistance profiling to evaluate bacterial strains' responses to antibiotics. The research targets commonly used water sources in the Barishal district, aiming to provide valuable insights for improved water quality management and public health interventions.

Each water sample was carefully collected to prevent contamination, employing sterile bottles and aseptic techniques (Gautam et al. 2021). After collection, the samples were immediately labeled, sealed, and transported to the laboratory for analysis. In order to prevent potential alterations in water samples, all samples were transported in an isolated foam container with ice, maintaining a temperature of 4–6 °C. All laboratory analyses were carried out at the Department of Microbiology and Public Health, Patuakhali Science and Technology University, Bangladesh.

To determine the concentration of coliform bacteria and assess the microbial potability of water samples, the most probable number (MPN) method was applied, as described by Alam et al. (2006). A total of 16 water samples, selected randomly from six distinct water sources, were aseptically collected and processed. Each sample was inoculated into lactose broth tubes prepared at three different strength conditions: five tubes with 0.1 mL (single strength), five tubes with 1 mL (single strength), and five tubes with 10 mL (double strength). The tubes were then incubated at 37 °C for 24 h. The presence of acid production and gas formation, indicated by color changes and bubble formation, respectively, was recorded as a positive result.

The statistical estimation of MPN was determined per 100 mL of the sample. Positive presumptive results were further confirmed by subculturing onto eosin methylene blue (EMB) agar, a selective and differential medium for the isolation of Escherichia coli. The absence of fecal coliforms in 100 mL of water was interpreted as compliance with potable water standards, thus classifying the sample as suitable for drinking.

The total viable count (TVC) was performed to quantify the overall bacterial load in the water samples. This analysis followed the ISO (1995) standard for microbiological methods. Water samples from each source were randomly selected, serially diluted, and distributed onto three Petri dishes containing plate count agar. The plates were incubated at 37 °C for 24 h, after which bacterial colonies were counted using a colony counter.

The colony-forming unit (CFU) per milliliter was calculated using the formula:

Calculation of CFU/mL:

CFU/mL = No of colonies × dilution factor × 1/aliquot (the volume of diluted specimen (aliquot) was either 0.1 or 1.0 mL).

Water samples that tested positive for gas production in the presumptive test were streaked onto EMB agar plates and incubated at 37 °C for 12 h. After incubation, colony morphology was examined to differentiate between coliform and non-coliform bacteria.

For further confirmation, a loopful of culture was inoculated into nutrient broth and incubated at 37 °C for 24 h under aerobic conditions. The enriched culture was then streaked onto MacConkey agar (MCA) plates and incubated at 37 °C for 24 h to assess its lactose fermentation characteristics.

Water samples were streaked onto mannitol salt agar (MSA) and MCA plates to facilitate the selective isolation of Staphylococcus spp. and NLFGN bacteria, respectively. The inoculated plates were incubated at 37 °C for 24 h under aerobic conditions. Following incubation, colony morphology was observed for preliminary identification. Gram staining was then performed to differentiate bacterial isolates based on their staining characteristics.

The bacterial isolates were identified using the standard microbiological methods from Bergey's Manual (Krieg et al. 2010). Moreover, the colonies were examined using the Gram staining method to identify their distinctive characteristics, including color, size, shape, surface texture, and opacity (Merchant & Packer 1967).

Bacterial isolates obtained from the water samples were subjected to antibiotic susceptibility testing using the Kirby–Bauer disk diffusion method (Hudzicki 2009). To determine the antibiotic sensitivity profile of the isolates, a total of 14 antibiotics such as neomycin (N 30), tobramycin (TOB 10), penicillin (P 10), gentamycin (GEN 10), oxytetracycline (O 30), amoxicillin (AMX 30), ampicillin (AMP 25), ceftriaxone (CTR 30), ciprofloxacin (CIP 5), enrofloxacin (EX 5), colistin (CL 10), levofloxacin (LE 5), erythromycin (E 15), and doxycycline (DO 30) were used. Zone diameters were measured and interpreted according to Clinical and Laboratory Standards Institute guidelines (CLSI 2005).

Descriptive statistics were calculated to summarize the findings. The obtained data were analyzed using Microsoft Excel (2010).

The visual representations in this study were generated using Canva, while the geographic mapping of the study area was conducted using ArcGIS (version 10.8, Esri, Redlands, CA, USA).

Table 1 presents the frequencies and percentages of water samples with TVC (CFU/mL) obtained from different water sources in the Barishal district, based on their accessibility and utilization. The majority of samples were collected from hotels (26.67%) and food vans (26.67%), followed by household tanks (20%), ponds (amounting to 13.32%), rivers, and farms (6.67%).

The water reserved in household tanks showed the highest degree of contamination with a mean TVC of 5.6 × 10⁵ CFU/mL, followed by hotel water (5.0 × 10⁵ CFU/mL), van water (4.8 × 10⁵ CFU/mL), river water (3.5 × 10⁵ CFU/mL), farm water (3.3 × 10⁵ CFU/mL), and pond water (3.2 × 10⁵ CFU/mL).

Table 2 summarizes the MPN results for 16 randomly selected water samples from six different water sources. Among the samples, 33.33% of Reserved Tank Water (RTW) samples were found to be microbiologically non-potable, highlighting significant contamination. Similarly, 20% of VW samples were non-potable, indicating risks associated with mobile water sources. In contrast, all samples from pond water (PW), river water (RW), farm water (FW), and hotel water (HW) were determined to be potable. Overall, 12.5% of the total water samples analyzed were classified as non-potable.

In this study, Staphylococcus spp. was present in all the sources of drinking water except pond water (Table 3). 50% of river and farm water samples were contaminated with Staphylococcus spp. followed by 33.33% tank water, 25% hotel water, and 12.5% van water (Table 3). Notably, E. coli was identified only in RTW (33.33%) and VW (25%) samples. Furthermore, NLFGN species were detected in PW (25%), hotel water (12.5%), and van water (12.5%).

Colonies with a characteristic green metallic sheen were observed on EMB agar, confirming the presence of E. coli (Figure S1a in the Supplementary Materials). The green metallic sheen is a distinctive feature used to identify coliform bacteria in water samples.

MSA supported the growth of Staphylococcus spp., which appeared as golden-yellow colonies (Figure S1c). This characteristic pigmentation is indicative of Staphylococcus aureus, commonly associated with potential health risks.

On MCA, NLFGN bacteria were identified by the appearance of colorless colonies, measuring approximately 2–3 mm in diameter (Figure S1b). The absence of pink coloration indicates an inability to ferment lactose.

The antibiogram status of bacterial isolates from various water sources revealed distinct patterns of AMR and susceptibility (Table 4).

E. coli isolates from RTW exhibited resistance to neomycin, doxycycline, ampicillin, penicillin, oxytetracycline, and amoxicillin. However, these isolates were susceptible to colistin and levofloxacin, while intermediate sensitivity was observed for gentamicin, tobramycin, and ceftriaxone. In contrast, E. coli isolates from VW showed resistance to doxycycline, ampicillin, penicillin, oxytetracycline, enrofloxacin, amoxicillin, colistin, and levofloxacin. These isolates were sensitive to ciprofloxacin, neomycin, erythromycin, gentamicin, tobramycin, and ceftriaxone.

Staphylococcus spp. isolates from RW demonstrated resistance to doxycycline, ampicillin, ciprofloxacin, penicillin, oxytetracycline, and amoxicillin. These isolates showed sensitivity to neomycin and levofloxacin, while intermediate responses were recorded for colistin, gentamicin, tobramycin, ceftriaxone, and enrofloxacin. Meanwhile, isolates obtained from FW were resistant to ampicillin, penicillin, oxytetracycline, amoxicillin, colistin, levofloxacin, and enrofloxacin. Sensitivity was noted for neomycin, ciprofloxacin, and erythromycin, with intermediate responses to doxycycline, gentamicin, and tobramycin.

For NLFGN bacterial isolates obtained from HW resistance to neomycin, doxycycline, ampicillin, penicillin, oxytetracycline, colistin, and amoxicillin was shown. These isolates were sensitive to levofloxacin and gentamicin, while ciprofloxacin, tobramycin, and ceftriaxone elicited intermediate responses. Similarly, NLFGN isolates from PW demonstrated resistance to ampicillin, penicillin, oxytetracycline, and amoxicillin. Sensitivity was observed for neomycin, ciprofloxacin, levofloxacin, doxycycline, gentamicin, tobramycin, and enrofloxacin, while intermediate sensitivity was noted for colistin and ceftriaxone.

These findings highlight the varying resistance and susceptibility patterns of E. coli, Staphylococcus spp., and NLFGN bacterial isolates from diverse water sources, emphasizing the potential risk of antibiotic resistance dissemination in the environment.

This study investigated the microbiological quality and AMR patterns of bacterial isolates from various water sources in Barishal, Bangladesh. The findings highlight substantial microbial contamination and the presence of antibiotic-resistant bacteria, raising significant public health concerns.

The TVC analysis revealed that RTW exhibited the highest microbial load (5.6 × 10⁵ CFU/mL), followed by HW and VW, with comparatively lower contamination observed in PW. These results are consistent with findings from studies conducted in urban areas of Bangladesh and other developing countries, which identified household water storage tanks and commercial water sources as significant reservoirs of microbial contamination due to inadequate maintenance and frequent human interaction, respectively (Chowdhury et al. 2014; Nabeela et al. 2014; Magar et al. 2019; Nishat et al. 2023). Research conducted in Dhaka city, Bangladesh, reported high levels of coliform bacteria (2.30 × 10⁵ CFU/mL) in household-stored water (Parveen et al. 2008). This high level of microbial contamination in stored water may be attributed to factors such as water stagnation and insufficient maintenance, as previously documented by Evison & Sunna (2001).

Furthermore, the MPN analysis indicated that 33.33% of the RTW samples were non-potable, suggesting substantial fecal contamination. This finding is consistent with Parvin et al. (2021), who highlighted the health risks associated with fecal contamination in household water sources, identifying it as a major contributor to gastrointestinal diseases, such as diarrhea, particularly among children in urban areas of Bangladesh. This emphasizes the need for regular tank cleaning, proper sealing, and disinfection.

Conversely, PW samples exhibited relatively lower contamination levels. This may be due to the natural purification processes in ponds, where beneficial microorganisms help break down organic matter, thereby improving water quality (Canadian Pond n.d.). However, seasonal factors, such as monsoonal runoff, may still elevate contamination risks in ponds, as highlighted by Alam et al. (2020). Therefore, proactive measures such as source protection and water treatment are essential to ensure long-term safety for human consumption.

In our study, Staphylococcus spp. and E. coli were the most frequently detected microorganisms across various water sources, which is consistent with previous research by Adesoji et al. (2019) in Nigeria and Khan & Bakar (2020) in Bangladesh. Both studies reported a high prevalence of Staphylococcus aureus (30%) and E. coli (62%) in water sources commonly consumed by humans. Additionally, the identification of NLFGN bacteria in HW, PW, and VW samples further supports the findings of Iwu et al. (2020), who observed the presence of such bacteria in water sources influenced by urban and hospital wastewater discharges. These results highlight a significant concern regarding water contamination in urbanized and hospital-adjacent areas. Furthermore, Kundu et al. (2018) emphasized that inadequate sanitation and hygiene practices are strongly associated with microbial contamination in drinking water. This reinforces the need for improved water management strategies to mitigate microbial risks and ensure safer drinking water.

Regarding the AMR patterns of the bacterial isolates, E. coli isolates from RTW and VW exhibited resistance to beta-lactam antibiotics, including ampicillin, penicillin, and amoxicillin. This finding is consistent with a study conducted in Dhaka, Bangladesh, which reported high levels of AMR in E. coli isolates from household water supplies (Talukdar et al. 2013). Despite these resistance patterns, we observed that the E. coli isolates from RTW retained susceptibility to colistin and levofloxacin. This aligns with recent observations suggesting that, although resistance is rising globally, certain antibiotics remain effective against environmental isolates.

However, the emergence of colistin resistance is a growing concern. Our study revealed colistin-resistant E. coli isolates, reflecting a broader trend seen in other regions. For instance, a study in Himachal Pradesh, India, reported the presence of colistin-resistant E. coli in water samples, marking a significant public health concern (Singh et al. 2021). Similarly, research in Lebanon identified multidrug-resistant E. coli isolates from irrigation water carrying the plasmid-mediated colistin resistance gene mcr-1, highlighting the role of water systems in the dissemination of AMR (Nasser et al. 2021). These findings underscore the need for ongoing surveillance and the development of effective antimicrobial stewardship strategies to mitigate the spread of resistant pathogens in water environments.

In our study, Staphylococcus spp. isolates from RW and FW exhibited extensive resistance to multiple antibiotics, including ampicillin, amoxicillin, penicillin, and oxytetracycline. This is concerning, as tap water from animal farms, which is used for both human and animal consumption, can serve as a significant vector for the spread of multidrug-resistant bacteria in both humans and farm animals. A report from Northeast Ohio documented Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus, in beach sand and freshwater sources (Thapaliya et al. 2017). The study suggested that human activity plays a major role in contaminating these environmental sites with multidrug-resistant Staphylococcus strains. Given that tap water from animal farms is shared for both agricultural and domestic purposes, the presence of resistant Staphylococcus spp. in these sources highlights the need for careful monitoring and management of AMR in water systems used by both humans and animals.

Similarly, NLFGN isolates from HW and PW exhibited resistance to beta-lactam antibiotics while maintaining susceptibility to aminoglycosides (gentamicin and tobramycin) and fluoroquinolones (ciprofloxacin and levofloxacin). This resistance profile aligns with findings from multiple studies on environmental Gram-negative bacteria. For instance, a study analyzing bacterial isolates from riverine drinking water sources reported that 81.8% of the isolates were multidrug-resistant, demonstrating resistance to a broad spectrum of antibiotics (Popoola et al. 2024). Furthermore, research on non-fermentative gram-negative bacteria in drinking water has emphasized their widespread distribution in the environment and their potential to cause a range of infections, particularly in immunocompromised individuals (Staradumskyte & Paulauskas 2014). These findings highlight the pervasive nature of AMR in environmental water systems and the potential health risks associated with exposure to resistant pathogens in these sources.

Strengths and limitations: This study presents several notable strengths that contribute to its scientific and public health significance. Notably, it represents the first comprehensive investigation of its kind in this region, providing essential insights into the microbiological quality of water and the associated AMR profiles of waterborne pathogens. By examining a broad spectrum of water sources – such as household reserved tank water, hotel water, street food van water, pond water, river water, and tap water from animal farms – the study enhances the external validity of its findings, making them applicable to a wide range of environmental contexts. Additionally, the in-depth AMR profiling of bacterial isolates offers valuable information on the prevalence of AMR in environmental settings, contributing to a pressing global health concern. Importantly, the study underscores the practical implications of its findings, advocating for improved water treatment protocols, sanitation practices, and the implementation of antibiotic stewardship strategies to mitigate potential public health risks.

Although this study has several strengths, it also has certain limitations that should be acknowledged. The most significant challenge encountered was financial constraints, which affected various aspects of the research process. Additionally, the relatively small sample size collected from each water source may restrict the generalizability of the findings to larger populations or different geographical regions. Another limitation of this study is that, while it successfully identifies bacterial isolates and AMR patterns, it does not include molecular identification and characterization. This has limited a deeper understanding of the specific bacterial strains and the underlying mechanisms driving resistance. In particular, the study was unable to identify the species or genus of NLFGN bacteria, as well as the specific strains or variants of Staphylococcus spp. Incorporating molecular techniques in future research could provide more precise taxonomic identification and enhance insights into the genetic determinants of resistance. Furthermore, the study does not account for seasonal variations, which are known to influence water quality and microbial load, potentially leading to the omission of important temporal trends. Lastly, a notable limitation of this study is the inability to assess AMR patterns for all antibiotics across all bacterial isolates. Additionally, TVC and MPN analyses could not be conducted for all samples, which may impact the comprehensiveness of the findings. Addressing these limitations in future research would enhance the robustness of the findings and improve their relevance for public health interventions.

Recommendations and future directions: Routine surveillance programs for water quality are essential to monitor microbial contamination and AMR patterns. These programs should integrate advanced molecular methods, such as Polymerase Chain Reaction (PCR) and whole-genome sequencing, to detect resistance genes and trace their sources. Further research should focus on the seasonal variation in microbial loads and AMR patterns in water sources to provide a comprehensive understanding of contamination dynamics. Expanding the scope to include molecular characterization of bacterial isolates will enable the identification of specific resistance mechanisms and enhance targeted interventions.

Public awareness campaigns emphasizing proper water handling, sanitation, and the prudent use of antibiotics are critical to reducing contamination risks and mitigating AMR dissemination. Strengthening collaboration among public health authorities, researchers, and community stakeholders will facilitate the development of effective water management policies and promote sustainable practices.

This study highlights significant microbial contamination and the presence of ARB in water sources across Barishal, Bangladesh, with the highest contamination levels observed in household reserved tank water. The study identified fecal pathogens, including E. coli and Staphylococcus spp., as well as widespread AMR, particularly against beta-lactam antibiotics. These findings emphasize the urgent need for improved water management, consistent monitoring, and effective water treatment interventions to safeguard public health. Further research is necessary to assess seasonal variations and the molecular mechanisms underlying resistance in environmental water sources.

This study was partially supported by the Research and Training Center (RTC) of Patuakhali Science and Technology University (Grant no. ANSVM 77, Fiscal year 2022–2023).

F.I.R. conceptualized the study, supervised the research, and revised the final draft. I.J.M. conducted the experiments and documented the results. A.S. performed the formal analysis, drafted the manuscript, generated figures, and created the maps. F.I.R. and M.E.H.K. provided critical reviews and contributed to the final revision of the manuscript. All authors approved the final manuscript.

During the preparation of this manuscript, the authors utilized ChatGPT (version 4.0) for linguistic improvements. The authors carefully reviewed and revised the content to ensure accuracy, completeness, and adherence to the journal's standards and take full responsibility for the final content of the published article.

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

The authors declare there is no conflict.