A review of technologies for rapid detection of …

1 A review of technologies for rapid detection of bacteriain recreational watersRachel T. Noble and Stephen B. WeisbergABSTRACTR achel T. Noble(corresponding author)University of North Carolina at Chapel Hill,Institute of Marine Sciences,3431 Arendell St, Morehead City,NC B. WeisbergSouthern California Coastal WaterResearch Project,7171 Fenwick Lane, Westminster,CA of recreational beaches for fecal indicator bacteria is currently performed using culture-based technology that can require more than a day for laboratory analysis, during which timeswimmers are at risk. Here we review new methods that have the potential to reduce themeasurement period to less than an hour.

2 These methods generally involve two steps. The first istarget capture, in which the microbial group of interest (or some molecular/chemical/orbiochemical signature of the group) is removed, tagged or amplified to differentiate it from theremaining material in the sample. We discuss three classes of capture methods: 1) Surface andwhole-cell recognition methods, including immunoassay techniques and molecule-specific probes;2) Nucleic acid methods, including polymerase chain reaction (PCR), quantitative PCR (Q-PCR),nucleic acid sequence based amplification (NASBA) and microarrays; and 3) Enzyme/substratemethods utilizing chromogenic or fluorogenic substrates.

3 The second step is detection , in whichoptical, electrochemical or piezoelectric technologies are used to quantify the captured, tagged oramplified material. The biggest technological hurdle for all of these methods is sensitivity, as EPA srecommended bathing water standard is less than one cell per ml and most detection technologiesmeasure sample volumes less than 1ml. This challenge is being overcome through addition ofpreconcentration or enrichment steps, which have the potential to boost sensitivity without theneed to develop new detector technology. The second hurdle is demonstrating a relationship tohealth risk, since most new methods are based on measuring cell structure without assessingviability and may not relate to current water quality standards that were developed inepidemiology studies using culture-based methods.

4 Enzyme/substrate methods may be the firstrapid methods adopted because they are based on the same capture technology as currently-approved EPA methods and their relationship to health risk can be established by demonstratingequivalency to existing procedures. Demonstration of equivalency may also be possible for somesurface and whole-cell recognition methods that capture bacteria in a potentially viable acid technologies are the most versatile, but measure nonviable structure and will requireinclusion in epidemiological studies to link their measurement with health words|E. coli, enterococci, water quality, recreational, optical, electrochemicalINTRODUCTIONC onsiderable resources are expended each year to measureindicator bacteria and assess whether recreational beachesare free from fecal contamination (Schiffet ).

5 Thesemonitoring programs are compromised, though, becausecurrent methods of enumerating bacteria are too slow toprovide full protection from exposure to waterbornepathogens. The current United States EnvironmentalProtection Agency (USEPA) approved methods to evaluatedoi: Publishing 2005 Journal of Water and Health| |2005recreational waters require an 18 to 96 hour incubationperiod, while several studies have shown that temporalchanges in indicator bacteria levels in beach water occur onmuch shorter time scales (Leecaster and Weisberg 2001;Boehmet ). Thus, contaminated beaches remainopen during the laboratory incubation period and are oftenclean by the time warnings are processing time lag can also negatively affecttracking of contamination sources.

6 A frequently usedtracking approach is to look for differential bacterialconcentrations at the confluence of upstream , the fecal contamination signal can dissipate ordisperse while the initial samples that would trigger such aninvestigation are being processed, making it difficult tosubsequently track the sources of fecal contamination. Evenwhen upstream tracking is successfully initiated, the slowlaboratory processing time requires that many locations beexamined simultaneously, rather than using spatially-sequential sampling that would be possible if a more rapid (and possibly field-based) method were limiting factor for present methods is that they relyon culturing techniques that either measure a metabolicendpoint or determine growth of a microorganism after anextended incubation period.

7 New molecular methods thatallow direct measurement of cellular properties withoutincubation are becoming available and have the potential toreduce the measurement period to less than an hour. Thesenew technologies also allow expansion of the number andtypes of microbiological indicators that can be monitoring programs are presently based primarilyonE. coliandEnterococcusspp. because they are easily andinexpensively cultured. Molecular methods do not requireculturing and allow for pathogens such as viruses to bemeasured as easily as bacterial indicators, potentiallyproviding a more direct link to public health development of molecular methods has advancedconsiderably for use in several disciplines, such as the foodservice and hospital industries (Fung 2002), there has been lesseffort toward application of new methods for recreationalwater qualitytesting.

8 Water testing canpresentchallenges thatare not frequently encountered in these other fields. Generallyspeaking, there are small numbers of the microorganisms ofinterest in a water sample, therefore large volumes of watermust typically be processed, or some enrichment or concen-tration approach included in sample processing. In addition,presence/absence information is not adequate for assessingrecreational waters. Methods must yield accurate quantitativeinformation. Other problems with water samples include thepresence of potential interferents to specific methodologicalapproaches, such as salinity, humic acids, highly variable andcomplex sample matrices, and the presence of other con-founding dominant native bacterial species.

9 In this paper, wereviewrapidmethodologiesthatarebeingde velopedforuseinrecreationalwatersandalso identifythemajorimpedimentstoadoption of these methods. For the purpose of this article, wetreat rapid as methods that provide results in less than 4hours (including sample preparation time), which is thelongest time frame that reasonably allows managers to takeactiontoprotectpublichealth( )onthesame day that water samples are OVERVIEWT here are two general steps involved in the application ofrapid technologies . The first is the capture, in whichthe microbial species or group of interest (or somemolecular/chemical/or biochemical signature of the group)is removed, tagged or amplified to differentiate it from theremaining material in the sample.

10 This step is typicallyresponsible for the selectivity of the approach. The secondstepisthedetection,inwhichthecaptu red,taggedoramplifiedmaterial is counted or measured quantitatively. The detectortypically acts as a transducer, translating the biological,physical, or chemical alteration into a measurable steps differ slightly among measurementapproaches, but they provide a useful outline for organizingour discussion. In many cases, a third step, preconcentration,may be added prior to target capture because most rec-reational waters have relatively dilute levels of contaminantscompared to other applications.