Enumeration of viable E. coli in rivers and wastewaters by fluorescent in situ hybridization

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Abstract

A combination of direct viable count (DVC) and fluorescent in situ hybridization (FISH) procedures was used to enumerate viable Escherichia coli in river waters and wastewaters. A probe specific for the 16S rRNA of E. coli labeled with the CY3 dye was used; enumeration of hybridized cells was performed by epifluorescence microscopy. Data showed that the method was able to accurately enumerate a minimum of 3000 viable E. coli among a large number of non-fecal bacteria. When applied to river water and wastewater samples, the DVC-FISH method gave systematically higher E. coli counts than a reference culture-based method (miniaturized MPN method). The ratio between both counts (DVC-FISH/MPN) increased with decreasing abundance of culturable E. coli indicating that the proportion of viable but non-culturable (VBNC) E. coli (detectable by the DVC-FISH procedure and not by a culture-based method) was higher in low contaminated environments. We hypothesized that the more stressing conditions, i.e. nutritional stress and sunlight effect, met in low contaminated environments were responsible for the larger fraction of VBNC E. coli. A survival experiment, in which sterile mineral water was inoculated with a pure E. coli strain and incubated, confirmed that stressing conditions induced the apparition of non-culturable E. coli detectable by the DVC-FISH procedure. The analysis of the E. coli concentration along a Seine river longitudinal profile downstream a large input of fecal bacteria by a WWTP outfall showed an increasing fraction of VBNC E. coli with increasing residence time of the E. coli in the river after release. These data suggest that the DVC-FISH method is useful tool to analyze the dynamics of fecal bacteria in river water.

Introduction

Freshwaters contaminated by fecal material from men and animals may contain a large variety of human pathogenic microorganisms. Health protection programs require to estimate the level of contamination of aquatic systems by these microorganisms. Since the systematic search of all potential strains of entero-pathogens is not feasible, the enumeration of various indicator bacteria is usually used to evaluate the fecal contamination of waters and, the sanitary risk associated with various utilization's of the water (bathing, production of drinking water, etc.). For years, total coliforms and fecal coliform were the most widely used indicators but, more recently, the abundance of Escherichia coli has been shown to be more related to the sanitary risk than that of coliforms (Fewtrell and Bartram, 2001).

Accurate enumeration of E. coli is thus important to assess microbiological water quality. Classical methods for enumerating E. coli are based on culture in liquid (most probable number, MPN) or solid (plate counts) media. These methods are time-consuming (usually a minimum of 24 h of incubation is needed) and do not allow to detect all the target bacteria in natural environments. Indeed, when released in natural waters, fecal bacteria were shown to loose their ability to grow on culture media while preserving their viability Colwell et al., 1985, Grimes and Colwell, 1986, Barcina et al., 1989, Pommepuy et al., 1996. The presence of these viable but non-culturable (VBNC) bacteria in the environment could be important from a sanitary point of view as some authors Colwell et al., 1985, Grimes and Colwell, 1986, Pommepuy et al., 1996 suggested that pathogenic VBNC bacteria could maintain their virulence being thus a potential reservoir of disease.

As a consequence, alternative methods were developed during the last 10 years to detect and enumerate E. coli in waters (Rompré et al., 2002). Those are direct enzymatic methods George et al., 2000, Van Poucke and Nelis, 2000, immunological methods (Pyle et al., 1999), quantitative polymerase chain reaction (PCR) (Juck et al., 1996) and fluorescent in situ hybridization (FISH) Regnault et al., 2000a, Lepeuple et al., 2003. This latter technique appears particularly interesting as it provides quantitative data in only 6–8 h (Rompré et al., 2002). Several fluorescently labeled DNA probes targeting E. coli were proposed in the literature Prescott and Fricker, 1999, Stender et al., 2001, Regnault et al., 2000a. Two major problems were encountered by the authors who used FISH for detecting fecal bacteria in natural waters: (i) the low level of targeted rRNA per cell due to the stress conditions met by these bacteria in such waters resulting in a low level of fluorescence and, thus, a difficult enumeration of the target events by epifluorescence microscopy or solid phase cytometry (Lepeuple et al., 2003); (ii) the inability to distinguish viable from non-viable cells.

To analyze simultaneously the phylogenetic affiliation and the metabolic state of single cells in waters, it is possible to couple FISH with methods characterizing the physiological state of bacteria at the single cell level as the direct viable count method (DVC) (Kogure et al., 1979), the CTC method (Rodriguez et al., 1992) or the esterase activity measurement (Catala et al., 1999). Regnault et al., 2000a, Regnault et al., 2000b proposed to couple the DVC procedure with the use of a FISH probe targeting the 16 s rRNA of E. coli. The DVC procedure involves exposing bacterial cells to a revification medium containing antibiotics preventing cellular division; elongated cells are then enumerated as viable cells. In the proposed protocol, the cells were hybridized after the revification stage with a 16S rRNA probe, called “Colinsitu”, specific from E. coli. The specificity of this probe for E. coli was fully demonstrated by Regnault et al., 2000a, Regnault et al., 2000b. The introduction of the revification stage allows, on one hand, to have in the viable elongated cells a sufficient rRNA content to be detectable by microscopy after the FISH procedure and, on the other hand, to distinguish viable E. coli from non-viable E. coli. Villarino et al. (2000) showed that the DVC-FISH procedure was adequate to monitor bacterial viability. A procedure combining DVC and FISH was also recently used to enumerate Enterobacteriaceae in natural and drinking waters (Baudart et al., 2002).

The aim of the present study was (i) to investigate the application of a protocol coupling DVC and FISH using the “Colinsitu” probe for enumerating by epifluorescence microscopy E. coli in river water and wastewater samples; (ii) to define the detection limit and the accuracy of this method; (iii) to compare the number of E. coli in rivers and wastewaters enumerated by the DVC-FISH procedure and by a culture-based method.

Section snippets

River and wastewater sampling

River water samples were collected from March to September 2003 in the Seine river hydrographical network (France). Samples were harvested from small rivers upstream to any domestic wastewater discharge up to the highly contaminated Seine river downstream to the Parisian area. Moreover, mean daily wastewater samples were collected at the entrance and at the outlet of nine wastewater treatment plants (WWTPs) located in France. The treatment in these plants included primary settling followed by

Accuracy and detection limits of the DVC-FISH procedure

To test the ability of the DVC-FISH procedure to enumerate low numbers of viable E. coli among a large abundance of non-target cells, variable amounts of E. coli were spiked in mineral water containing a constant 1.5×108 non-E. coli bacteria/100 ml.

The abundance measured by the DVC-FISH method was compared to the abundance estimated by the MPN method (Fig. 1); no significant difference (Mann–Whitney U-test) was found between counts performed by both methods. The relative error (in %) was

Discussion

The search for more rapid, sensitive and specific methods for the enumeration of bacterial indicators of fecal contamination resulted in the development of numerous alternatives to the classical culture methods during the recent years (Rompré et al., 2002). Apart from the fact that they require 24–48 h to obtain a result, the major limitation of traditional culture-based methods lies in their inability to detect VBNC bacteria. In this work, a combination of the DVC and the FISH procedures was

Conclusions

Our results demonstrated that an epifluorescence microscopic DVC-FISH procedure can be used to enumerate viable E. coli in rivers and wastewaters. This method, on the contrary to the culture-based methods, allows to obtain results within a working day but is presently too time consuming to be considered for routine microbiological water analysis. It can nevertheless be recommended as a very useful research tool to understand the processes involved in the dynamics of E. coli in the environment.

Acknowledgements

Tamara Garcia Armisen benefits from a doctoral grant from the “Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture” (FRIA) (Belgium). This study was a part of the PIREN Seine program of the Centre National de la Recherche Scientifique (France). The authors thank Adriana Anzil and Philippe Mercier for their help during the field work, and Karine Delabre, Philippe Lebaron and Julia Baudart for helpful discussions during the course of the study.

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