7 Real-time monitoring technologies for indicator bacteria and pathogens in shellfish and shellfish harvesting waters A.P. Dufour and G.N. Stelma Jr. The counting of bacteria in water has been acriticalelement in protecting public health in the last century. In the early part of the century scientists were able to grow many species of bacteria and differentiate them from each other using biochemical tests. They also had observed that certain bacteria were always found in the faeces of humans and otherwarm-blooded animals and that significant disease was alsoassociated with faecal wastes. This association was recognized early on by the scientists who were the fore-runners of our present-day public health scientists. Abacterium originally called Bacillus coli # 2010 World Health Organization (WHO). Safe Management of Shellfish and Harvest Waters. Edited by G. Rees, K. Pond, D. Kay, J. Bartram and J. Santo Domingo. ISBN: 9781843392255. Published by IWA Publishing, London, UK. 110 Safe Management of Shellfish and HarvestWaters was found in high numbers in the faeces of an infant and shortly thereafter was found in the faeces of healthy humans and warm-blooded animals (Escherich 1885). Pathogens, such as the microorganism that causes cholera, were difficult to grow with the nutrient media available at the time. B. coli,on the otherhand, grew on simple media and was easily detected in water. The specific identification of B. coli., however, was not easy and microbiologists were soon isolatingall of the bacteria that lookedlike and behaved like B. coli. This resulted in the group name coliform, meaninghaving the form of a B. coli. Soon, glucose was replacedincoliform media with lactose. The ability of coliforms to specifically metabolize lactoseand produceacid and hydrogen gas as end-products allowed this group of organisms to be easily identified. Dunham (1898) recognized the valueofgas production for detecting coliforms and used this characteristic to identify the presence of coliforms in liquid culture. He simply inverted asmall tube and placed it in the culturetube. This tube capturedthe gas produced by coliforms. Phelps (1907) developed a coliform indexwhich used utilized gas production. The indexwas based on adilution concept in which the reciprocal of the highest dilution where growth and gas production occurred was reported as the best estimate of the coliform density in agiven volume of water. McCrady (1915) was the first to utilize statistical probability theory to estimatenumbers of coliform bacteria using the fermentation tube method. The technique, based on the number of tubesshowing growth and gas production, provided amostprobable number (MPN)estimateofthe number of coliform bacteria in aparticularvolumeofsample (McCrady 1915). However, solid media, to which water samples can be applied, were preferable to MPN methods because colonies growing on the surface of the medium couldbe counted rather than estimated. Initially the small volumes of sample that must be used with solid agar media precluded their use with samplesthat contain small numbers of the bacteria being counted. This problem was solved with the introduction of the membrane filter which allowed larger volumes of water to be analyzed.100 ml or moreofsample can be passed through the membrane filter, which can then be placed on aselective, solid medium and incubated for aselected time period, after which coloniesonthe filter can be identified and counted. The common factor betweenthese twoquantitative approaches to counting bacteria is the requirement for cell multiplication to occur over asufficient period of time, so they can be easily observed. In both cases the time to colony formation is between20and 24 hours.Compressing this time interval has been the goal of water microbiologists for manyyears. In one case this was accomplished usingamembrane filter technique and anutrient medium that Real-timemonitoring technologies 111 maximized the cell doubling time which allowed microscopic identification of the colonies in about seven hours (Reasoner et al. 1979). This is about the lowest time limit for obtaining results with culturemethods for quantifying bacterial indicatorsinwater samples. However, there is anew generation of instruments and techniques being developed to quantifyindicator bacteria and pathogens that will provide monitoring results in amuchshorter period of time. Molecular methods are at the forefrontofthese new technologies. The advent of the polymerase chain reaction (PCR) technique (Mullis and Faloona 1987) holds great promiseasarapid method for measuring environmental water quality.The PCR was asignificant step in the development of highly specific, rapid methods for identifying microbes associated with faeces. It did have drawbacks, however,inthat the original post-amplification procedures were time consuming and did not lend themselves to quantification. In the mid- 1990’s,the real-time quantitative PCR (qPCR) was introduced to the scientific community (Heid et al. 1996). This procedure allows both detection and quantification of PCR-amplified nucleic acidsequences without the need for post-amplificationprocedures. There are numerous thermal cycling instruments available commercially that are capable of rapidly detecting and quantifying microbes in environmental watersbythis technique. Other techniques are also gaining favour in the area of water quality monitoring. Chemicalmethods that measureadenosine triphosphate (ATP) and enzymatic reactions are also rapid and may be useful for measuring water quality.Antibody based methods that are used with flowcytometry and fibre optic technologiesalso have some potential, but problems with sensitivity and the small volumes used in these assays are limiting their use. Molecular methods however,are by far the most advanced of the technologies that have been used to quantify microbes used to measurefaecal contamination in water. This chapter will describesome of the available technology for the rapid measurement of water quality and shellfish. Although many high technology methods are described in the literature, very few have been used to test natural samples, such as water samples or shellfishtissue samples. Only methods that have been used to measurenatural water samples, whethermarine, estuarine or freshwater, or shellfishtissuesfor faecal indicator bacteria or pathogens will be described in this chapter.The approach will be to describethe procedure in somedetail and then briefly review one or two papersfrom the literature describing how the method has been used to measureindicatorsorpathogens in samplestaken from natural environmental watersorfrom harvested shellfish. No attempt has been made to provide acomprehensive literature review. 112 Safe Management of Shellfish and HarvestWaters 7.1 MOLECULAR APPROACH The most studied of the new methods for quantifyingmicrobes in water is the qPCR. This technologyhas many attributes which makeitattractive for measuring microbes in water. First, the qPCR method is very specifictothe target microbes beingdetected. Contemporary culturetechniques depend on phenotypic characteristics whosepresence may be governed by several enzymes that frequently are affected by the physiologicalstate of the microbes. A variablephysiological state will result in variablephenotypiccharacteristics which can at timesmakeidentification of the microbe difficult. This variability does not occur with qPCR which detects cells on the basis of specific nucleotide sequences that are uniquetothe microbes under study. In addition, the qPCR technology is very rapid. Detecting and identifying microbes with cultural methods usually require about 24 hours,the amount of time it takesmicrobes to grow to the point where growth can be visualized. qPCR results, on the other hand, can be observed in two to three hours,because of the logarithmic amplification of the sequences of interest. The qPCR process consists of two steps that occur at different temperatures. At ahigh temperature, double-stranded DNA is denaturedtotwo single strands, completing the first step. At the lower temperature, anumber of reactions take place. The first is the hybridization of short pieces of DNA (oligonucleotides) calledprimers to specific locations on the single strand of DNA. These primers provide astarting point for the synthesis of new double-stranded DNA.Asecond hybridization involving ahighly specific oligonucleotide called aprobe, takes place at apoint on one of the single-strandsofDNA which is betweenthe two primer sites. This probe is unique to the microbe beingdetected. One of the most commonly used types of probesiscalled ahydrolysis of Taqman1 probe. These probeshave afluorescent reporter dye attached to one end and aquencher dye attached to the other end. When these two dyes remain in close proximity to each other on the probe the reporter dye cannot fluoresce. After the probe attaches to the target sequence, apolymerase begins extending the primer toward the probe,forming new double-stranded DNA. As the extended DNA meets the probe,the probe is cleaved, freeing the reporter dye so that it is no longer in proximity to the quencher dye and can now fluoresce. The formation of double- strandedDNA completely removes the probe from the target sequence allowing the primer extensiontocontinue until anew double-stranded DNA is formed, ending the secondstep. These cycles are programmedinto aspectrofluorometric thermocycler,which continuously proceeds through the two steps, measuring the amount of fluorescent dye freed in each annealing step. Thefluorescent