**2. Established analytical methods to detect** *E. coli* **in environmental waters**

particulate matter followed by a filtration step; the bacteria retained on the membrane are incubated on a growth medium for up to 18 h. *E. coli* are selected on colony colour and identified using chromogenic agar, confirmation can be via API 20E strips or PCR. Some strains of *E. coli* which are β‐D‐glucuronidase negative, such as *E. coli* O157:H7, will not be detected as *E. coli* but, as they are β‐D‐galactosidase positive, they will appear as coliform bacteria on selected chromogenic agars [29]. A range of chromogenic agars are available for the detection of *E. coli* O157:H7 which have improved specificity when compared to cefixime‐tellurite Sorbitol MacConkey (CT‐SMAC) [30] when tested against eight environ-

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Amirat *et al*. [32] used the membrane filtration procedure followed by culture on selective chromogenic media to monitor bacterial contamination of the river Thames. This procedure successfully identified *Salmonella, Enterococci, Klebsiella pneumoniae* and *E. coli*. Sixty percent of the samples were in excess of the EU standard for bathing water and the study demonstrated frequent sewage pollution of the Thames which was most noticeable after heavy rainfall. The relationship between sewage contamination of rivers and heavy rainfall has also been reported in other studies: Tryland *et al*. [33] used the Colifast early warning system while Kolarevic *et al*. [34] studied the river Tisa in India using the membrane filtration method. The MPN method was also used to demonstrate an increase in indicator organisms including *E. coli* 2 days after rainfall in the river Göta Älv in Sweden [35] and to measure faecal pollution and antibiotic resistance in the river Cauvery in India [36]. Faecal contamination of the river Danube was measured using the indicator organisms: coliforms and *Enterococci* [37]. A rapid onsite method for *E. coli*, ColiSense, is based on the direct fluorescent analysis of β‐D‐glucuronidase activity in recreational water samples [38]. Total time taken to complete the analysis was approximately 103 minutes of which 75 minutes were needed to complete the assay. The total time taken to obtain a result depends on the time to transport the sample from the test site to the laboratory and time for any pre-treatment steps. This method does offer greater rapidity and portability but there may be differences in the results obtained with

Monitoring of environmental water samples is usually carried out using culture-based faecal indicators of microbial contamination. However, these methods are expensive and time‐ consuming and recently efforts have been made to develop methods which give more rapid results at lower cost and greater specificity. Indirect detection of *E. coli* and total coliforms in water samples from Canadian fresh water beaches using a portable detection system has been described [39]. The detection procedure was based on the fluorescent detection of β‐Dglucuronidase and β‐D-glucuronidase using novel anthracene-based enzyme substrate. The method is able to detect single cells of either *E. coli* or total coliforms within 18 h and turbidity and colour and turbidity of the water samples does not affect the result. False‐positive coliform results due to the presence of *Aeromonas spp.* could be eliminated by the inclusion of

A number of semi-automated systems are currently available which utilise selective growth media and fluorometric substrates. The Colifast Analyser system utilises 4‐methylumbelliferone‐β‐D‐ glucuronide to detect *E. coli* and 4‐nitrophenyl‐β‐D‐galactoside to detect coliforms using defined

mental samples inoculated with *E. coli* [31].

this procedure than with standard culture procedures.

Cefsulodin in the growth medium.

Methods involving culturing procedures are essentially laboratory based and, although they are sensitive, usually involve one or two days before the result is known. They can be used to detect the presence of a range of potential contaminating organisms in addition to the primary target organism. Culture procedures rely on either biochemical, immunological or molecular methods to identify the bacteria present. However, culture methods may underestimate the bacterial load or fail to grow relevant organisms as they measure only the viable organisms present in the samples that can be cultured. In environmental samples, a significant number of cells may not be detected despite being viable. Viable but non-culturable cells (VBNC) result from stress encountered in the environment or the condition and content of the samples [21]. Therefore, alternative new technologies that do not rely on growing the bacteria in culture are required; many of these involve nucleic acid based methods [6]. Chromogenic agar can detect non‐growing cells by measuring the presence of an enzyme e.g. β‐D‐galactosidase for coliforms [22] and β‐D‐glucuronidase for *E. coli* [23]. A wide range of media is available for the characterisation of environmental microorganisms [24]. Detailed descriptions of standard laboratory procedures which are used in environmental studies including microscopic as well as biochemical characterisation are given in Alexander and Strete [25]. The rapid identification of known bacteria can be achieved using the API® ID Strip Range (BioMerieux, France) which consists of a series of miniaturised techniques based on established laboratory procedures.

The reference methods for detection and isolation of *E. coli* and coliforms in water are the membrane filtration method (ISO 9308‐1:2014) and the multiple tube fermentation (Most Probable Number, MPN, ISO 9308‐2:2012). ISO 9308‐1:2014 is based on membrane filtration and subsequent culture on a chromogenic coliform‐agar medium [26]. Due to the low selectivity of the differential agar medium, background growth can interfere with the reliable enumeration of *E. coli* and coliform bacteria, for example, in surface waters or shallow well waters. This method is not suitable for these types of water. As the MPN method (ISO 9308‐2:2012) is based on the growth of the target organisms in liquid medium it is suitable for most waters but should not be used for enumeration of bacteria in marine samples as dilution of the sample is required. A recent study compared membrane filtration (MF) and multiple tube fermentation (MTF) procedures to analyse water obtained from a dockside and a beach in California [27]. The MF method gave more reliable and precise data than the MTF method. The later method was more time consuming, labour intensive and less precise. The MF procedure also has the advantage of being able to examine large volumes of water but it has limitations when dealing with turbid water samples. The *E. coli* and coliform content in water samples from five Environmental Protection Agency regions (EPA) in the USA were compared using the ColilertTM automated test and MTF procedure [28]. Similar results were obtained with both methods; however, the ColilertTM procedure was easier to perform and interpret.

Enumeration and characterisation of bacteria in environmental samples requires a tiered approach. The samples collected from, e.g., rivers are diluted or centrifuged to remove particulate matter followed by a filtration step; the bacteria retained on the membrane are incubated on a growth medium for up to 18 h. *E. coli* are selected on colony colour and identified using chromogenic agar, confirmation can be via API 20E strips or PCR. Some strains of *E. coli* which are β‐D‐glucuronidase negative, such as *E. coli* O157:H7, will not be detected as *E. coli* but, as they are β‐D‐galactosidase positive, they will appear as coliform bacteria on selected chromogenic agars [29]. A range of chromogenic agars are available for the detection of *E. coli* O157:H7 which have improved specificity when compared to cefixime‐tellurite Sorbitol MacConkey (CT‐SMAC) [30] when tested against eight environmental samples inoculated with *E. coli* [31].

**2. Established analytical methods to detect** *E. coli* **in environmental** 

128 *Escherichia coli* Escherichia coli - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications - Recent Advances on Physiology, Pathogenesis and Biotechnological Applications

Methods involving culturing procedures are essentially laboratory based and, although they are sensitive, usually involve one or two days before the result is known. They can be used to detect the presence of a range of potential contaminating organisms in addition to the primary target organism. Culture procedures rely on either biochemical, immunological or molecular methods to identify the bacteria present. However, culture methods may underestimate the bacterial load or fail to grow relevant organisms as they measure only the viable organisms present in the samples that can be cultured. In environmental samples, a significant number of cells may not be detected despite being viable. Viable but non-culturable cells (VBNC) result from stress encountered in the environment or the condition and content of the samples [21]. Therefore, alternative new technologies that do not rely on growing the bacteria in culture are required; many of these involve nucleic acid based methods [6]. Chromogenic agar can detect non‐growing cells by measuring the presence of an enzyme e.g. β‐D‐galactosidase for coliforms [22] and β‐D‐glucuronidase for *E. coli* [23]. A wide range of media is available for the characterisation of environmental microorganisms [24]. Detailed descriptions of standard laboratory procedures which are used in environmental studies including microscopic as well as biochemical characterisation are given in Alexander and Strete [25]. The rapid identification of known bacteria can be achieved using the API® ID Strip Range (BioMerieux, France) which consists of a series of miniaturised techniques based on established

The reference methods for detection and isolation of *E. coli* and coliforms in water are the membrane filtration method (ISO 9308‐1:2014) and the multiple tube fermentation (Most Probable Number, MPN, ISO 9308‐2:2012). ISO 9308‐1:2014 is based on membrane filtration and subsequent culture on a chromogenic coliform‐agar medium [26]. Due to the low selectivity of the differential agar medium, background growth can interfere with the reliable enumeration of *E. coli* and coliform bacteria, for example, in surface waters or shallow well waters. This method is not suitable for these types of water. As the MPN method (ISO 9308‐2:2012) is based on the growth of the target organisms in liquid medium it is suitable for most waters but should not be used for enumeration of bacteria in marine samples as dilution of the sample is required. A recent study compared membrane filtration (MF) and multiple tube fermentation (MTF) procedures to analyse water obtained from a dockside and a beach in California [27]. The MF method gave more reliable and precise data than the MTF method. The later method was more time consuming, labour intensive and less precise. The MF procedure also has the advantage of being able to examine large volumes of water but it has limitations when dealing with turbid water samples. The *E. coli* and coliform content in water samples from five Environmental Protection Agency regions (EPA) in the USA were compared using the ColilertTM automated test and MTF procedure [28]. Similar results were obtained with both

methods; however, the ColilertTM procedure was easier to perform and interpret.

Enumeration and characterisation of bacteria in environmental samples requires a tiered approach. The samples collected from, e.g., rivers are diluted or centrifuged to remove

**waters**

laboratory procedures.

Amirat *et al*. [32] used the membrane filtration procedure followed by culture on selective chromogenic media to monitor bacterial contamination of the river Thames. This procedure successfully identified *Salmonella, Enterococci, Klebsiella pneumoniae* and *E. coli*. Sixty percent of the samples were in excess of the EU standard for bathing water and the study demonstrated frequent sewage pollution of the Thames which was most noticeable after heavy rainfall. The relationship between sewage contamination of rivers and heavy rainfall has also been reported in other studies: Tryland *et al*. [33] used the Colifast early warning system while Kolarevic *et al*. [34] studied the river Tisa in India using the membrane filtration method. The MPN method was also used to demonstrate an increase in indicator organisms including *E. coli* 2 days after rainfall in the river Göta Älv in Sweden [35] and to measure faecal pollution and antibiotic resistance in the river Cauvery in India [36]. Faecal contamination of the river Danube was measured using the indicator organisms: coliforms and *Enterococci* [37]. A rapid onsite method for *E. coli*, ColiSense, is based on the direct fluorescent analysis of β‐D‐glucuronidase activity in recreational water samples [38]. Total time taken to complete the analysis was approximately 103 minutes of which 75 minutes were needed to complete the assay. The total time taken to obtain a result depends on the time to transport the sample from the test site to the laboratory and time for any pre-treatment steps. This method does offer greater rapidity and portability but there may be differences in the results obtained with this procedure than with standard culture procedures.

Monitoring of environmental water samples is usually carried out using culture-based faecal indicators of microbial contamination. However, these methods are expensive and time‐ consuming and recently efforts have been made to develop methods which give more rapid results at lower cost and greater specificity. Indirect detection of *E. coli* and total coliforms in water samples from Canadian fresh water beaches using a portable detection system has been described [39]. The detection procedure was based on the fluorescent detection of β‐Dglucuronidase and β‐D-glucuronidase using novel anthracene-based enzyme substrate. The method is able to detect single cells of either *E. coli* or total coliforms within 18 h and turbidity and colour and turbidity of the water samples does not affect the result. False‐positive coliform results due to the presence of *Aeromonas spp.* could be eliminated by the inclusion of Cefsulodin in the growth medium.

A number of semi-automated systems are currently available which utilise selective growth media and fluorometric substrates. The Colifast Analyser system utilises 4‐methylumbelliferone‐β‐D‐ glucuronide to detect *E. coli* and 4‐nitrophenyl‐β‐D‐galactoside to detect coliforms using defined substrate technology which is used for online monitoring. The endpoints are yellow for total coliforms and fluorescent for *E. coli*. There is also a micro hand held version available. Results can be obtained using this procedure within 2–12 h. An alternative system, Colilert® 3000 (Seres, France) utilises fluorescent or chromogenic substrates and can deliver results within 24 h. These methods correlate well with standard laboratory methods although the results were two to three orders of magnitude higher than MTF and MPN methods probably due to the presence of *Aeromonas spp.* and *Vibrio spp.* (natural inhabitants of the surface water) known to interfere with the Colilert test [40]. A comprehensive study by Schang [41] compared four methods to analyse riverine, estuarine and marine environments near Melbourne, Australia. They compared the industry‐standard IDEXX (Colilert®) culture‐based method with three alternative approaches: the TECTATM automated system uses fluorometric assays [42] and while still under development they found a good correlation between the IDEXX and TECTATM procedures while the later had the advantage of a faster turnaround time. Good correlation was found between the IDEXX method and the US EPA Method 1611 for qPCR detection of *Enterococci*. Good correlation was found between next‐generation‐sequencing (NGS) and the culture‐based procedures; however, the cost of NGS is too high at present, but future developments might make the use of this procedure suitable for routine screening.

system for the *in situ* detection of faecal indicator bacteria [48] showing the future potential for

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Recent advances in sequencing technology and the decrease in costs for whole genome sequencing have made this technology the forefront of investigations into outbreaks of infectious diseases and food or water contamination [49–51]. Rapid identification can be achieved and the outbreak quickly be traced to its source allowing for more effective treatment and containment. This provides an entirely new and effective tool that allows tracing a faecal contamination of water to its source. Measures can then be put in place to contain the current release, prevent future events and if the cause is found to be a careless or deliberate release, legal proceeding can be initiated. However, for routine monitoring of water quality this technology is not a viable alternative as it is more expensive, requires specialist equipment and

The coupling of microarray technology with PCR enhances detection and identification of bacterial contaminants in water samples. Several commercial kits are now available for the assay of *shiga* toxin producing *E. coli* O157:H7 in environmental samples. More recently, detection techniques using biosensors have shown potential for onsite monitoring. These combine a rapid biochemical reaction with a physicochemical signal that is proportional to the concentration of the target molecule and thus the number of bacteria present in a sample. The biomarkers targeted are most commonly the enzymes established in laboratory‐based assays. We have shown that a direct assay of 1 ml river water sample for β‐D-glucuronidase activity analysed with a portable fluorimeter can achieve detection limits of 7 cfu/ml within 30 min [52], the ColiSense system described by Heery [38] combines incubation and fluorescent detection in a portable device achieving below 100 cfu/ml in 75 min and a recent study by Hesari [53] describes a biosensor, sensitive enough for the detection of *E. coli* in drinking water with a significant fluorescent signal generated in under 2 h and no sample processing. Wutor [54] describes a biosensor targeting β‐D-galactosidase that can detect 1 cfu/100 ml in 15 min using voltammetry to detect the enzyme activity. A system that combines concentration of *E. coli* with a colorimetric detection of enzyme activity and is easy to use, portable and not requiring any instrumentation was recently developed and commercialised [55]. Several immunosensors have also been developed, mostly in order to detect specific bacterial antigens correlated with virulence. A detection limit of 100 cfu/ml is achieved by a specific immunosensor for *E. coli* O157:H7 [56], and with a gold-nanoparticle sensor described more recently, *E. coli* O157:H7 were detected as low as 10 cfu/ml in 1 h [57]. An electrochemical biosensor capable of specifically detecting ESBL *E. coli* strains was developed and achieved a detection limit of 5000 cfu/ml [58]. A third type of biosensors targets nucleic acids and Paniel [59] has shown that both optical and electrochemical detection methods can achieve detection limits below 20 cfu/ml *E. coli* in seawater. Capacitors can be utilised to detect whole cells and a recent paper describes a biosensor that can specifically detect *E. coli* to a limit of 70 cfu/ml in river water by combining a capacitive biosensor with microcontact imprinting [60]. A number of different biosensor systems for the detection of bacteria in water and studies evaluating these are

bringing molecular analysis out of the laboratory and constructing robotic analysers.

trained analysts and does not provide rapid or onsite results.

reviewed by Lopez‐Roldan [61].

The use of indicator organisms is well established and will probably continue as the gold standard of microbial contamination until reliable alternative procedures are developed. There are however several promising areas of development which are considered in the sections below which provide valuable supplementary information and have the potential to evolve in specific easy to use onsite procedures. Culture procedures take a minimum of 24 h to complete and the availability of more rapid techniques will allow earlier appropriate management decisions to be made.
