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Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system.

ABSTRACT: 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 10(5) 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 10(3) active AOB ml(-1) were detected in the membrane-intact fraction, and 1.40 x 10(4) 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.

SUBMITTER: Hoefel D

PROVIDER: S-EPMC1287627 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system.

Hoefel Daniel D Monis Paul T PT Grooby Warwick L WL Andrews Stuart S Saint Christopher P CP

Applied and environmental microbiology 20051101 11

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 d ...[more]

PMID: 16269672

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Project description:BACKGROUND:Residual disinfection is often used to suppress biological growth in drinking water distribution systems (DWDSs), but not without undesirable side effects. In this study, water-main biofilms, drinking water, and bacteria under corrosion tubercles were analyzed from a chloraminated DWDS (USA) and a no-residual DWDS (Norway). Using quantitative real-time PCR, we quantified bacterial 16S rRNA genes and ammonia monooxygenase genes (amoA) of Nitrosomonas oligotropha and ammonia-oxidizing archaea-organisms that may contribute to chloramine loss. PCR-amplified 16S rRNA genes were sequenced to assess community taxa and diversity. RESULTS:The chloraminated DWDS had lower biofilm biomass (P=1×10-6) but higher N. oligotropha-like amoA genes (P=2×10-7) than the no-residual DWDS (medians =4.7×104 and 1.1×103amoA copies cm-2, chloraminated and no residual, respectively); archaeal amoA genes were only detected in the no-residual DWDS (median =2.8×104 copies cm-2). Unlike the no-residual DWDS, biofilms in the chloraminated DWDS had lower within-sample diversity than the corresponding drinking water (P<1×10-4). Chloramine was also associated with biofilms dominated by the genera, Mycobacterium and Nitrosomonas (?91.7% and ?39.6% of sequences, respectively). Under-tubercle communities from both systems contained corrosion-associated taxa, especially Desulfovibrio spp. (?98.4% of sequences). CONCLUSIONS:Although residual chloramine appeared to decrease biofilm biomass and alpha diversity as intended, it selected for environmental mycobacteria and Nitrosomonas oligotropha-taxa that may pose water quality challenges. Drinking water contained common freshwater plankton and did not resemble corresponding biofilm communities in either DWDS; monitoring of tap water alone may therefore miss significant constituents of the DWDS microbiome. Corrosion-associated Desulfovibrio spp. were observed under tubercles in both systems but were particularly dominant in the chloraminated DWDS, possibly due to the addition of sulfate from the coagulant alum.

Project description:The diagnosis and management of pneumonia are limited by the use of culture-based techniques of microbial identification, which may fail to identify unculturable, fastidious, and metabolically active viable but unculturable bacteria. Novel high-throughput culture-independent techniques hold promise but have not been systematically compared to conventional culture. We analyzed 46 clinically obtained bronchoalveolar lavage (BAL) fluid specimens from symptomatic and asymptomatic lung transplant recipients both by culture (using a clinical microbiology laboratory protocol) and by bacterial 16S rRNA gene pyrosequencing. Bacteria were identified in 44 of 46 (95.7%) BAL fluid specimens by culture-independent sequencing, significantly more than the number of specimens in which bacteria were detected (37 of 46, 80.4%, P ≤ 0.05) or "pathogen" species reported (18 of 46, 39.1%, P ≤ 0.0001) via culture. Identification of bacteria by culture was positively associated with culture-independent indices of infection (total bacterial DNA burden and low bacterial community diversity) (P ≤ 0.01). In BAL fluid specimens with no culture growth, the amount of bacterial DNA was greater than that in reagent and rinse controls, and communities were markedly dominated by select Gammaproteobacteria, notably Escherichia species and Pseudomonas fluorescens. Culture growth above the threshold of 10(4) CFU/ml was correlated with increased bacterial DNA burden (P < 0.01), decreased community diversity (P < 0.05), and increased relative abundance of Pseudomonas aeruginosa (P < 0.001). We present two case studies in which culture-independent techniques identified a respiratory pathogen missed by culture and clarified whether a cultured "oral flora" species represented a state of acute infection. In summary, we found that bacterial culture of BAL fluid is largely effective in discriminating acute infection from its absence and identified some specific limitations of BAL fluid culture in the diagnosis of pneumonia. We report the first correlation of quantitative BAL fluid culture results with culture-independent evidence of infection.

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Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system.

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Culture-independent techniques for rapid detection of bacteria associated with loss of chloramine residual in a drinking water system.

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