Methods for detection and enumeration of coliforms in drinking water: a review

Safe drinking water is essential for maintaining good health and is a fundamental right of all human beings. Infectious diseases transmitted through water impact around 1.8 billion individuals on a global scale and contribute to over 3 million fatalities annually due to diarrheal complications. Children below the age of 5 are particularly susceptible to waterborne illnesses as a result of ingesting contaminated drinking water (WHO 2016). Water that has been contaminated could potentially harbour a significant concentration of pathogenic microorganisms, presenting a substantial threat to human well-being. Typically, water samples undergo examination for the existence of microorganisms to verify the safety of the drinking water. Numerous potential pathogens could be associated with water, rendering it impractical to comprehensively examine water samples for every pathogen. Moreover, the methods available for pathogen detection in water exhibit limited sensitivity and need larger sample volumes for accurate analysis due to relatively low pathogen concentrations in water. A specialized laboratory along with highly trained manpower and appropriate biosafety containment is essential for the detection of pathogens. In addition, the process of identifying and confirming the presence of pathogens in water is time consuming and can take several days. As a result, indicator organisms have been used for assessing faecal contamination in drinking water.

Coliforms within the Enterobacteriaceae family are frequently employed as an indicator of water quality, particularly for drinking water. This diverse group includes genera such as Escherichia, Citrobacter, Enterobacter, and Klebsiella. Coliforms are prevalent in the intestines of humans and other warm-blooded animals, as well as in various natural environments like soil. However, the survival of coliforms is not typical within drinking water settings (Maheux et al. 2014). Coliform bacteria are preferred as indicator organisms because they can be readily detected, and the methods employed for enumerating them are relatively cost-effective. They also exhibit a strong correlation with the presence of other waterborne pathogens, including viruses, protozoa, and various other bacteria. Coliforms have served as indicator organisms for many years and are integral components of regulatory standards governing drinking water and recreational water quality. Their presence in treated drinking water can signify issues such as ineffective disinfection and treatment, regrowth problems in the water supply system, or the inadvertent introduction of faecal contaminants into potable water distribution systems (Clark et al. 1996).

Conventional techniques employed for the identification and quantification of coliforms rely on the culturing of microorganisms using selective solid or liquid media. These well-established approaches include the multiple-tube fermentation (MTF) technique, membrane filtration (MF), and plating methods, such as the spread plate and pour plate techniques (Maheux et al. 2014). In the past three decades, innovative fluorogenic and chromogenic substrates have been developed and utilized for the specific and rapid quantification of coliforms. These classical culture-based methods have gradually given way to more advanced immunological assays and molecular techniques, including enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). These modern methods enable the detection of coliforms within a shorter timeframe, typically taking only 3–4 h, in contrast to culture-based methods, which necessitate a 24-h incubation period (Maheux et al. 2014). A comprehensive overview of the various techniques available for coliform enumeration is provided in the subsequent section.

The MTF technique, also recognized as the most probable number (MPN) method, is one of the earliest well-established and universally accepted standard procedures for semi-quantitative coliform enumeration in potable water and wastewater (APHA Method 9221 1992). This method employs a statistical approach to estimate the most probable coliform counts, relying on the capacity of coliform bacteria to ferment lactose and produce acid and gas within specified timeframes. The MTF method is characterized by its simplicity, cost-effectiveness, and applicability to a wide array of sample types. In addition, it facilitates the recovery and growth of injured organisms and ensures easy result interpretation. Nonetheless, the precision of this method depends on factors such as the number of tubes employed for water sample analysis, the choice of culture medium, and incubation conditions (Rompré et al. 2002). Several factors can contribute to an underestimation of coliform counts when utilizing this technique, including media inhibitory properties and potential interference from non-coliform bacteria in high numbers (McFeters et al. 1982). While the specificity of the MTF technique is generally satisfactory, it is not infallible. Some non-coliform bacteria possess the ability to ferment lactose, leading to occasional false-positive outcomes. To mitigate this limitation, the MTF method is typically followed by confirmatory tests, encompassing the differentiation of coliform bacteria via cultural, biochemical, and serological methods.

The MF method represents an extensively employed and accepted standard method (APHA Method 9222 1992; USEPA Method 1604 2002) for detecting and quantifying coliform bacteria within the water and other environmental samples. The fundamental principle behind this method is filtration, wherein the sample undergoes filtration through a sterile membrane, eliminating larger particles while capturing bacteria on the membrane's surface. The bacteria retained on the filter are subsequently retrieved by placing the membrane onto a selective and differential medium. It is frequently necessary to dilute the sample before filtration due to the variable concentration of coliforms in natural water samples. Typically, a range of dilutions is prepared to prevent overcrowding and ensure that at least one plate contains a countable colony number. The number of required dilutions depends on the anticipated coliform concentration and the sensitivity of the employed culture medium.

Various culture media and incubation conditions have been followed for the isolation of coliforms from water samples using this method. Commonly employed media for analysing drinking water using this method include membrane-Endo (m-Endo) media, Tergitol-Triphenyl Tetrazolium Chloride Tergitol (TTC) media, MacConkey agar, and Teepol media (Grabow & du Preez 1979). To enumerate faecal coliforms, the membrane Fecal Coliform Agar medium (an enriched lactose medium) is employed, with incubation typically carried out at a higher temperature of 44.5 °C for 24 h (APHA 1998). Furthermore, some MF media have been developed, incorporating chromogenic substrates to facilitate the detection of total coliforms and E. coli in water. Examples include MI agar (BD, Franklin Lakes, NJ, USA) (Brenner et al. 1993) and m-ColiBlue (Hach, Loveland, CO, USA) media (Grant 1997). One of the major concerns with this method is its inability to recover injured or stressed coliforms (McFeters et al. 1982). Further, the presence of a substantial number of heterotrophic bacteria may decrease the recovery of coliform bacteria (Burlingame et al. 1984).

Microorganisms are typically clumped and not distributed evenly throughout the distribution system. In such scenarios, large numbers of samples collected from the distribution system must undergo testing because the likelihood of detecting faecal bacteria in only a few samples is considerably low. The presence/absence test provides a better alternative for routine and frequent monitoring of water quality due to its simplicity, cost-effectiveness, and ability to analyse numerous samples quickly (Clark 1990). Several commercially available P/A test kits are used for coliform enumeration, including the H₂S test kit, Colilert-18, Colisure, Colisense, m-Coliblue, Aquagenx test, etc. (USEPA 2019). The use of P/A tests effectively eliminates many of the errors associated with more complex enumeration techniques. It does not demand sophisticated equipment or extensive analytical expertise, making it user friendly and simple to implement.

The most commonly used presence/absence test kit in developing countries is the hydrogen sulphide (H₂S) test kit (Manja et al. 1982). This test relies on detecting H₂S gas produced by bacteria associated with faecal contamination such as Citrobacter, Clostridium perfringes, Salmonella, Proteus, and others. Many modified versions of the H₂S test have emerged demonstrating a strong correlation between the H₂S test and traditional MPN and MF methods for detecting faecal contamination in drinking water (McMahan et al. 2011). Nevertheless, there are several limitations associated with the H₂S test including false-positive results. This can occur due to the production of H₂S by non-faecal bacteria from natural sources (Sobsey & Pfaender 2002). Bacterial H₂S production can be attributed to the reduction of sulphate and other oxidized forms of sulphur, as well as the degradation of sulphur-containing amino acids and other organic compounds present in biomass (Mosley & Sharp 2005). Due to these limitations, this test may not be suitable for specific indicators of microbiological parameters such as E. coli. As an alternative, a presence/absence test kit (^MColiPAT) was developed by Tambi et al. (2020) that can detect low levels of coliforms within a shorter incubation period, typically 8–10 h.

Enzymatic methods rely on specific enzymatic activities exhibited by coliform bacteria, such as β-glucuronidase or β-galactosidase, to identify their presence in water samples. ß-d-galactosidase has been used for the isolation of total coliforms which catalyze the conversion of lactose into glucose and galactose. On the other hand, ß-d-glucuronidase catalyzes the hydrolysis of ß-d-glucopyranosiduronic derivatives into their corresponding aglycons and d-glucuronic acid. This enzymatic activity is primarily associated with E. coli within the Enterobacteriaceae family. However, it is less prevalent in other members of the same family, such as Shigella, Yersinia, and Salmonella (Frampton & Restaino 1993).

Edberg & Edberg (1988) introduced ‘defined substrate methods’ as an innovative approach for isolating coliforms and E. coli to overcome the limitations associated with MF and MTF techniques. These methods use specific substrates such as O-nitrophenyl-β-d-galactopyranoside (ONPG) to detect the enzyme ß-d-galactosidase (present in total coliforms) and 4-methylumbelliferyl-β-d-glucuronide (MUGlu) to detect the enzyme ß-glucuronidase (for specific detection of E. coli). One of the first commercially available tests utilizing this approach was Colilert-18, manufactured by IDEXX Laboratories, United States of America (USA). The Colilert method demonstrates a strong correlation with the conventional MPN and MF methods and is also accepted as a standard test for the identification of coliforms in water samples (APHA Method 9223 1992). Originally designed as a presence/absence test, it can be used for quantitative enumeration also using the multitube technique. The United States Environmental Protection Agency (USEPA) has approved various enzyme-based methods for the identification and isolation of indicator microbes including Colilert^®, Enterolert^®, Colisure^® (IDEXX Laboratories, Portland, ME, USA) m-ColiBlue24^® (Hach, Loveland, CO, USA), Readycult^®, Chromocult^® (EMD Millipore, Burlington, MA, USA), and E*Colite (Charm Sciences Inc, Lawrence, MA, USA) (USEPA 2019). Several chromogenic and fluorogenic substrates used for the rapid and accurate detection or enumeration of coliforms are summarized in Table 1.

Enzymatic approaches present numerous advantages in comparison to conventional culture-based methods. These benefits include enhanced specificity, high sensitivity, the capacity to distinguish between various coliform types, and expedited outcomes. However, these methods are more expensive and require specialized equipment, making them less accessible to many water quality monitoring programs. Frequently, a hybrid approach employing both enzymatic and culture-based methods is adopted to achieve a more comprehensive evaluation of water quality.

Microorganisms can be directly detected through the labelling of organisms or their components with fluorescent labels such as fluorescein, which can be detected and quantified through the use of epifluorescence microscopy or laser scanning. The epifluorescence microscopy technique is quite labour intensive and time consuming and therefore has been replaced with the laser scanning technique. The ChemScanRDI^® instrument (Chemunex, France) has been specifically designed for the quick identification and quantification of microorganisms that have been fluorescently labelled (Rompré et al. 2002). In this approach, microorganisms are first filtered through membrane filters, and subsequently, they are tagged with fluorescent probes. These labelled microorganisms can then be scanned using a laser. The generated signals are subjected to a series of computer analyses that distinguish between labelled organisms and fluorescent debris. Epifluorescence microscope can be used simultaneously for visual validation of results. This technique has been reported to detect E. coli, Cryptosporidium oocysts, and Giardia cysts after labelling with a fluorescently labelled monoclonal antibody and a fluorescently labelled 16S rRNA probe. Some of the laser scanning devices reported earlier are summarized in Table 2.

Laser scanning is a promising approach for the identification of coliforms in water samples. It offers faster results, higher sensitivity, and high specificity, and can detect a wider range of bacteria compared to traditional culturing methods. However, this method is still being developed and validated and may not be suitable for all types of coliforms and sample matrices. Further research is needed to validate the method and to make it more accessible and affordable for routine use.

Molecular methods have been devised to shorten analysis times while maintaining high sensitivity and specificity obviating the necessity for confirmatory steps and cultivation processes. Moreover, these methods are capable of detecting viable but non-culturable bacteria in water samples in a short period. The following section discusses the molecular methods reported for detecting coliforms in drinking water.

These approaches rely on employing specialized antibodies designed for the precise recognition of antigens found on the surfaces of the target microorganisms. Both monoclonal and polyclonal antibodies have been reported for the detection of indicator bacteria in water samples (Law et al. 2015). A pre-cultivation step is essential for such methods to eliminate the detection of dead cells and to increase the sensitivity of the method. Immunological methods based on antigen–antibody reactions can be performed using various approaches like the ELISA technique (ELISA), immunofluorescence assay (IFA), and immunomagnetic separation (IMS).

The ELISA method is simple, rapid, and highly sensitive capable of detecting antigenic protein levels as low as 10⁻⁹ g. Despite these attributes, the utilization of ELISA assays for coliform enumeration from natural environments has been somewhat limited, primarily due to a range of challenges, including the non-specific binding of antigens (Hanai et al. 1997). The specificity of these methods is dependent on the type and concentration of the antibody used. In addition, interference from diverse materials and non-target heterotrophic microorganisms within the sample further reduces the specificity of these methods.

IFA can be used for the identification and enumeration of a single cell in water samples collected from natural habitats (Campbell 1993). This technique involves the coupling of fluorochrome with a specific antibody designed to target indicator bacteria's antigen. The fluorescently labelled cells were then detected through the use of flow cytometry or by epifluorescence microscopy (Karo et al. 2008). An alternative approach for detecting viable bacteria combines immunofluorescence with a respiratory activity compound. This method has been effectively employed in the identification of Salmonella typhimurium, E. coli O157: H7, and Klebsiella pneumonia in water (Pyle et al. 1995).

The IMS technique offers another viable approach for the rapid assessment of microorganisms. This method entails concentrating and purifying the target microorganism through the use of magnetic beads coated with either polyclonal or monoclonal antibodies. The sample is gently mixed with immunomagnetic beads and then subjected to magnetic separation using a designated magnet. This process holds the target microorganisms against a recovery vial, allowing the removal of non-bound material. Subsequently, the isolated target organisms can be cultured or directly identified after the removal of magnetic beads through vortexing. This technique has demonstrated its efficacy in identifying minimal quantities of microorganisms, including E. coli O157:H7, within food and water samples (Deshmukh et al. 2016).

The immunological methods are simple and rapid but have several limitations. The specificity and affinity of monoclonal antibodies determine the efficacy of immunological methods. However, the method's specificity can be compromised due to the potential cross-reactivity of antibodies with closely related antigens found in various bacterial strains. Moreover, elevated turbidity in water samples can interfere with the detection and enumeration of target organisms. A significant constraint arises from the absence of antibodies against E. coli strains isolated from environmental sources, limiting the wider application of immunological techniques for E. coli detection. Complex investigations are required for the identification of monoclonal antibodies for coliforms. As a result of these constraints, immunological techniques have encountered challenges in effectively detecting coliforms including E. coli in drinking water.

These methods are based on the fundamental principle of detecting specific DNA or RNA sequences in the target microorganism. This involves the pairing of a particular DNA or RNA sequence with a synthetic oligonucleotide (Ramírez-Castillo et al. 2015). Hybridization can take place either between the DNA probe and the target DNA sequence or between the DNA probe and the rRNA sequence. Importantly, these methods do not necessitate the culturing of microorganisms and, as a result, can identify target microorganisms within a few hours. Several nucleic acid-based methods have been developed for the quantification of indicator bacteria, including FISH, PCR, multiplex PCR (mPCR), real-time PCR, microarrays, and pyrosequencing.

This method relies on the capacity of nucleic acid to hybridize with specific complementary nucleic acid sequences present in a target microorganism. Specific DNA or RNA probes labelled with fluorescent markers targeted against 16S rRNA sequence present within microorganisms are used in this approach. Flow cytometry or fluorescence microscopy can be used for the detection of specific microbial populations based on the degree of complementary matching between the target sequence and the probe (Amann & Fuchs 2008). This method has been used to enumerate pathogens, coliforms, and Enterococci in water samples. Some of the oligonucleotide probes used for identifying members of the Enterobacteriaceae family including E. coli are ENT1 (Loge et al. 1999), COLINSINTU (Regnault et al. 2000), and peptide nucleic acid (PNA) probe (Prescott & Fricker 1999). In addition, this method has also been used for detecting viable but not culturable (VBNC) microorganisms using a direct viable count assay (Wingender & Flemming 2011).

In this method, a target DNA fragment is amplified primers in vitro using oligonucleotide primers catalyzed by a DNA polymerase enzyme (Taq polymerase) (Maheux et al. 2011). This amplification process occurs over several cycles to ensure sufficient multiplication of the target DNA sequence. The amplified DNA sequences are subsequently detected through agarose gel electrophoresis. Notably, this technique can identify an extremely low number of indicator microorganisms within a brief time frame. PCR technique has been used for the enumeration of various waterborne pathogens and indicator microorganisms such as E. coli, Clostridium perfringens, and so on. However, due to the diverse genera present in the coliform group, no single primer can be universally used for the detection of coliforms in water. The primers targeting the lacZ gene for the identification of commonly found enzyme ß-galactosidase within the coliforms were used earlier for the enumeration of coliforms (Bej et al. 1990, 1991). However, the lacZ-based primers are inadequate for specifically determining coliforms since they cannot differentiate between Serratia odorifera and Hafnia alvei (Fricker & Fricker 1994). Various genes have been identified for designing primers to achieve the specific detection of E. coli in water. These include lamB gene (Bej et al. 1990), uidA and uidR genes (Bej et al. 1991), and phoE gene (Spierings et al. 1993). Among them, the uidA and uidR genes that encode for the enzyme ß-glucuronidase were reported to be the most successful one for the specific detection of E. coli (Juck et al. 1996). Multiplex and real-time PCR variants have gained widespread adoption for quantifying coliforms within drinking water.

mPCR allows for the simultaneous detection of multiple gene targets, potentially enhancing the specificity of the PCR method. This method is more rapid compared to the conventional PCR technique. One of the significant requirements in this assay is designing multiple primers that must have the same annealing temperature for a successful assay (Omar & Barnard 2014). The successful amplification of the target nucleic acid sequence in mPCR can be influenced by various factors including the primer length, PCR buffer concentration, Taq polymerase, DNA template, and magnesium chloride (Omar & Barnard 2014).

Real-time PCR allows for the rapid detection of microorganisms as well as the quantitative evaluation of amplified DNA fragments by measuring fluorescent signals emitted by labelled probes or intercalating dyes (Girones et al. 2010). The intensity of the fluorescence is directly proportional to the concentration of PCR products. In clinical microbiology, this technique has been used for enterotoxigenic E. coli strains (Bellin et al. 2001). Several commonly used fluorescent systems for real-time PCR include Taqman probes, SYBR green, molecular beacons (Deshmukh et al. 2016), and the CRENAME method (Maheux et al. 2011).

An important drawback of the PCR method is its limited capacity to analyse only a small volume of a sample. This constraint necessitates the consideration of a pre-concentration step to ensure the effective enumeration of microorganisms. However, certain inhibitory substances like iron and humic acids present in natural water may also get concentrated along with nucleic acid which may reduce the specificity of the assay. Another limitation is that this assay cannot differentiate between live and dead microorganisms as the nucleic acid sequence may be released from the dead microbe in the water (Wang et al. 2009). The use of 3–4 h of pre-incubation of the sample in a selective medium is required to differentiate between viable and non-viable cells (Frahm et al. 1998). However, this pre-incubation step may further enhance the cost and time for the detection of the target organism. Most of the research work using PCR methods has been carried out using pure bacterial culture strains instead of direct environmental samples. There are additional challenges associated with the application of PCR-based methods for enumerating microorganisms from environmental samples, such as the inhibition of the Taq polymerase enzyme and the bonding of target DNA with colloidal matter (Holcomb & Stewart 2020).

The choice of methods for detecting and enumerating coliforms will depend on multiple considerations including the type of sample, the degree of coliform contamination, the requisite level of sensitivity, available resources, and the desired turnaround time. Therefore, it is always a good idea to carefully evaluate the options and choose the method that best fits the specific needs and constraints of the situation at hand. The strengths and limitations of various methods for the detection and quantification of coliforms in water are presented in Table 3, which may facilitate the final selection.

The latest progress in molecular biology, molecular recognition elements, and nanomaterials has expanded the potential for highly specific and sensitive detection of coliforms. Innovative approaches such as loop-mediated isothermal amplification (LAMP), paper-based microfluidic devices and biosensors, phage-based assays, and nanoparticles have emerged enabling rapid, specific, and real-time monitoring. LAMP targets specific DNA sequences of coliform bacteria, and its isothermal nature makes it conducive to field applications, enabling onsite detection without the requirement for specialized equipment (Lin et al. 2018; Manzanas et al. 2023). Paper-based microfluidic devices have emerged as versatile platforms for coliform detection. These devices integrate microfluidic channels and molecular probes on paper substrates, allowing capillary action to drive sample flow and reagent mixing (Snyder et al. 2020). They are cost-effective, portable, and amenable to mass production (Noviana et al. 2021). Paper-based sensors including dipstick tests and lateral flow assays leverage antibodies, enzymes, and other molecular probes to capture coliform bacteria and yield visual or smartphone-readable results (Ali et al. 2017). These biosensors convert biological interactions into measurable electrical or optical signals, leading to the rapid detection of coliforms at a very low cost (Gunda et al. 2017). Phage-based assays consist of engineered phages carrying reporter genes that can infect coliforms and produce detectable bioluminescence signals (Hinkley et al. 2018; Alonzo et al. 2022). These assays offer high specificity and allow for the discrimination of viable coliform cells from non-viable ones (Hussain et al. 2021). Nanoparticles functionalized with antibodies selectively bind to bacteria, amplifying the signal for detection (Shanker et al. 2020). Furthermore, the integration of artificial intelligence (AI) and machine learning streamlines data analysis, allowing for automated interpretation and pattern recognition and ultimately enhancing accuracy and efficiency in coliform detection (Wang et al. 2020; Kotwal et al. 2022). These platforms offer rapid, specific, and quantitative results, paving the way for real-time monitoring.

Although coliform bacteria have traditionally served as vital indicators of water quality, it is essential to recognize their inherent limitations in the context of safeguarding public health and safety. One primary concern lies in their non-specificity as coliforms can be found in non-faecal environments, leading to potential misinterpretation of contamination sources (Saxena et al. 2015). Furthermore, coliform tests are inadequate for detecting the potential presence of certain waterborne pathogens such as viruses (e.g., norovirus, rotavirus) and parasitic protozoans (e.g., Cryptosporidium, Giardia), raising concerns about underestimating waterborne disease risks (Payment & Locas 2011). It has also been reported that coliform bacteria can form biofilms on surfaces in water distribution systems, which can protect them from disinfection processes and make their detection in water samples challenging (Kilb et al. 2003). In addition, recent findings also indicate that relying solely on coliform enumeration to determine the disinfectant dosage may not be sufficient to guarantee effective disinfection, as various opportunistic pathogens, including coliforms, can proliferate following regeneration, reactivation, and regrowth after disinfection (Shekhawat et al. 2021). These limitations underscore the need for a comprehensive water quality assessment that incorporates multiple indicators, including specific pathogen testing, chemical parameters, and risk assessment methodologies, aligning with evolving regulatory standards.

Coliform bacteria serve as important indicators of water quality and the presence of potential faecal contamination, making their accurate detection and quantification crucial for ensuring public health and safety. This review article has highlighted the various traditional and advanced techniques available for the detection and enumeration of coliforms in water. Traditional methods such as the MTF and MF methods have long been employed and approved as standard methods by the USEPA due to their simplicity and reliability (USEPA 2019). However, these methods are time consuming and require a significant amount of manual labour. Molecular-based methods such as PCR and quantitative PCR (qPCR) offer rapid and sensitive detection of coliforms. These methods target specific genetic markers and enable the identification and enumeration of coliforms with high precision. While these methods provide numerous advantages, their implementation may be limited by cost, technical expertise, and infrastructure requirements. Therefore, careful consideration of factors such as sensitivity, specificity, speed, cost-effectiveness, feasibility, and specific needs is essential for the selection of an appropriate method for coliform detection. In addition, the establishment of standardized protocols and quality control measures is crucial to ensure the accuracy and comparability of results across different laboratories. Further, coliforms' persistence in the environment, susceptibility to regrowth, and sensitivity to environmental factors complicate their utility as sole indicators. Recognizing these constraints, modern water quality monitoring practices increasingly incorporate a broader array of parameters, including specific pathogen testing and physicochemical analyses to offer a more comprehensive assessment of water safety.

For effective, targeted, and economically viable assessment of water quality, advanced molecular techniques have also emerged as game-changers for coliform detection. LAMP, paper-based microfluidic devices, phage-based assays, nanoparticles, and biosensors are collectively shaping a future where water quality assessment is rapid, accessible, and efficient. Microfluidic systems enable the manipulation of small volumes of liquids, making them ideal for miniaturized and rapid detection methods. Bacteriophage-based biosensors and LAMP offer rapid results and cost-effective detection, with potential applications in water quality monitoring. Nanoparticles such as gold nanoparticles and magnetic nanoparticles enhance the sensitivity of detection methods by specifically binding to coliforms, amplifying the signal and allowing for detection even at low concentrations. Advanced data analysis techniques, including machine learning and AI, assist in processing large datasets generated by various detection methods. Collectively, these advanced technologies represent a paradigm shift in water quality assessment, holding immense potential to transform the approach towards coliform detection and water quality assessment.

The financial support for this study was provided by the Water and Sanitation Support Organization (WSSO), PHED, Jaipur, Government of Rajasthan, India (Sanction No. WSSO/WQ/2016-2017/4794, dated 16.01.2017).

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

The authors declare there is no conflict.