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Applied and Environmental Microbiology, November 2005, p. 6479-6488, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6479-6488.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Culture-Independent Techniques for Rapid Detection of Bacteria Associated with Loss of Chloramine Residual in a Drinking Water System

Daniel Hoefel,1,2* Paul T. Monis,1,2 Warwick L. Grooby,1 Stuart Andrews,2 and Christopher P. Saint1,2

The Cooperative Research Centre for Water Quality and Treatment, Australian Water Quality Centre, SA Water Corporation, Salisbury, South Australia 5108, Australia,1 School of Pharmacy and Medical Sciences, Division of Health Sciences, University of South Australia, City East Campus, Adelaide, South Australia 5001, Australia2

Received 9 February 2005/ Accepted 13 June 2005

Chloramination is often the disinfection regimen of choice for extended drinking water systems. However, this process is prone to instability due to the growth of nitrifying bacteria. This is the first study to use alternative approaches for rapid investigation of chloraminated drinking water system instability in which flow cytometric cell sorting of bacteria with intact membranes (membrane-intact fraction) (BacLight kit) or with active esterases (esterase-active fraction) (carboxyfluorescein diacetate) was combined with 16S rRNA gene-directed PCR and denaturing gradient gel electrophoresis (DGGE). No active bacteria were detected when water left the water treatment plant (WTP), but 12 km downstream the chloramine residual had diminished and the level of active bacteria in the bulk water had increased to more than 1 x 105 bacteria ml–1. The bacterial diversity in the system was represented by six major DGGE bands for the membrane-intact fraction and 10 major DGGE bands for the esterase-active fraction. PCR targeting of the 16S rRNA gene of chemolithotrophic ammonia-oxidizing bacteria (AOB) and subsequent DGGE and DNA sequence analysis revealed the presence of an active Nitrosospira-related species and Nitrosomonas cryotolerans in the system, but no AOB were detected in the associated WTP. The abundance of active AOB was then determined by quantitative real-time PCR (qPCR) targeting the amoA gene; 3.43 x 103 active AOB ml–1 were detected in the membrane-intact fraction, and 1.40 x 104 active AOB ml–1 were detected in the esterase-active fraction. These values were several orders of magnitude greater than the 2.5 AOB ml–1 detected using a routine liquid most-probable-number assay. Culture-independent techniques described here, in combination with existing chemical indicators, should allow the water industry to obtain more comprehensive data with which to make informed decisions regarding remedial action that may be required either prior to or during an instability event.

* Corresponding author. Mailing address: Australian Water Quality Centre, SA Water Corporation, Private Mail Bag 3, Salisbury, South Australia 5108, Australia. Phone: 618 8259 0321. Fax: 618 8259 0228. E-mail: daniel.hoefel{at}sawater.com.au.

Applied and Environmental Microbiology, November 2005, p. 6479-6488, Vol. 71, No. 11
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.11.6479-6488.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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