**3. Molecular techniques for bacterial identification in environmental waters**

Molecular techniques for the specific detection and quantification of bacteria are highly sensitive, rapid and specific, they can be readily automated and standardised so have some advantages over the standard culture-based techniques. Detection does not rely on the target organisms being viable and multiply under culture conditions or on the expression and activity of enzymes or other biochemical markers. However, where low numbers of bacteria are present, an enrichment step is often required limiting the aforementioned advantages. Quantitative PCR methods provide accurate numbers of genomes present and multiplex approaches can simultaneously identify the target organism and test for genes associated with pathogenicity or antibiotic resistance [43, 44] and host‐specific detection thus linking the contamination to a source [45–47]. Both sample clean-up and PCR protocols have recently been developed to be fast and simplified and requiring a limited set of laboratory resources thus making molecular analysis a more attractive option for routine monitoring and even field testing. The development of automated DNA extraction and PCR methods have been utilised to develop an autonomous system for the *in situ* detection of faecal indicator bacteria [48] showing the future potential for bringing molecular analysis out of the laboratory and constructing robotic analysers.

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

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

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

**3. Molecular techniques for bacterial identification in environmental** 

Molecular techniques for the specific detection and quantification of bacteria are highly sensitive, rapid and specific, they can be readily automated and standardised so have some advantages over the standard culture-based techniques. Detection does not rely on the target organisms being viable and multiply under culture conditions or on the expression and activity of enzymes or other biochemical markers. However, where low numbers of bacteria are present, an enrichment step is often required limiting the aforementioned advantages. Quantitative PCR methods provide accurate numbers of genomes present and multiplex approaches can simultaneously identify the target organism and test for genes associated with pathogenicity or antibiotic resistance [43, 44] and host‐specific detection thus linking the contamination to a source [45–47]. Both sample clean-up and PCR protocols have recently been developed to be fast and simplified and requiring a limited set of laboratory resources thus making molecular analysis a more attractive option for routine monitoring and even field testing. The development of automated DNA extraction and PCR methods have been utilised to develop an autonomous

this procedure suitable for routine screening.

decisions to be made.

**waters**

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 trained analysts and does not provide rapid or onsite results.

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 reviewed by Lopez‐Roldan [61].

Proteomics methods have been developed and extensive databases created allowing the identification of microorganisms directly in complex samples. Several studies have shown how MALDI‐TOF‐MS (Matrix‐Assisted Laser Desorption Ionisation Time‐of‐flight Mass Spectrometry) can be employed to identify organisms at species level, and detect virulence and resistance markers in environmental waters [62, 63]. A study by Loff [64] compares proteomics analysis with molecular and biochemical methods for the detection of microorganisms commonly associated with water safety. It can be expected that future developments of this technology will widen its application in many diagnostic and analytical applications.

with further developments of NGS and MALDI. However, the advantage of the currently used *E. coli–*based procedures is their simplicity, low cost and functionality for rapid onsite detection. Additional more broad ranging tests would need to be rigorously assessed in a wide variety of environmental situations before they could be adapted as international standards. There is, therefore, a clear need to re‐examine the precision and reproducibility of both culture and molecular-based methods in the assessment of environmental samples to take into account local variations and design new methods to be applicable for a wide range of scenarios in order to make a significant contribution to improving water safety globally.

*E. coli* as an Indicator of Contamination and Health Risk in Environmental Waters

http://dx.doi.org/10.5772/67330

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Sensitive and frequent monitoring of environmental waters is essential to minimise adverse effect on human health. The current approach to monitoring for contamination in environ-

**Figure 1.** Current approach to monitoring and identifying bacteria in environmental water.

**5. Conclusions**

mental waters is shown in **Figure 1**.

It has to be noted that the identification of organisms and detection of virulence or resistance by both molecular and proteomics approaches relies on the comparison of results with existing databases. This limits to the identification of known strains and characterised genes and proteins and is thus unlikely to achieve detection of uncultivable microorganisms. However, a combination of recent advances in bioinformatics and novel methods like the one described by Kaeberlein [65] have increased our knowledge about the microbial world and extended our database resources. Molecular and proteomics methods have shown great potential in the identification in temporal and special distribution of microorganisms in the aquatic environment and to combine species identification with detection of virulence and drug resistance. Future developments are likely to combine the best of both worlds to achieve robust assessment of water quality by quantifying indicator organisms to detect contamination and identify virulence and resistance markers to assess public health risks and inform stakeholders on the need and nature of required interventions.
