Iron uptake is essential for Escherichia coli survival in drinking water

AIMS: The aim of this study was to elucidate if the need for iron for Escherichia coli to remain cultivable in a poorly nutritive medium such as the drinking water uses the iron transport system via the siderophores. METHODS AND RESULTS: Environmental strains of E. coli (isolated from a drinking water network), referenced strains of E. coli and mutants deficient in TonB, an essential protein for iron(III) acquisition, were incubated for 3 weeks at 25 degrees C, in sterile drinking water with and without lepidocrocite (gamma-FeOOH), an insoluble iron corrosion product. Only cells with a functional iron transport system were able to survive throughout the weeks. CONCLUSIONS: The iron transport system via protein TonB plays an essential role on the survival of E. coli in a weakly nutritive medium like drinking water. SIGNIFICANCE AND IMPACTS OF THE STUDY: Iron is a key parameter involved in coliform persistence in drinking water distribution systems.     

Detection of Escherichia coli in biofilms from pipe samples and coupons in drinking water distribution networks

Fluorescence in situ hybridization (FISH) was used for direct detection of Escherichia coli on pipe surfaces and coupons in drinking water distribution networks. Old cast iron main pipes were removed from water distribution networks in France, England, Portugal, and Latvia, and E. coli was analyzed in the biofilm. In addition, 44 flat coupons made of cast iron, polyvinyl chloride, or stainless steel were placed into and continuously exposed to water on 15 locations of 6 distribution networks in France and Latvia and examined after 1 to 6 months exposure to the drinking water. In order to increase the signal intensity, a peptide nucleic acid (PNA) 15-mer probe was used in the FISH screening for the presence or absence of E. coli on the surface of pipes and coupons, thus reducing occasional problems of autofluorescence and low fluorescence of the labeled bacteria. For comparison, cells were removed from the surfaces and examined with culture-based or enzymatic (detection of beta-d-glucuronidase) methods. An additional verification was made by using PCR. Culture method indicated presence of E. coli in one of five pipes, whereas all pipes were positive with the FISH methods. E. coli was detected in 56% of the coupons using PNA FISH, but no E. coli was detected using culture or enzymatic methods. PCR analyses confirmed the presence of E. coli in samples that were negative according to culture-based and enzymatic methods. The viability of E. coli cells in the samples was demonstrated by the cell elongation after resuscitation in low-nutrient medium supplemented with pipemidic acid, suggesting that the cells were present in an active but nonculturable state, unable to grow on agar media. E. coli contributed to ca. 0.001 to 0.1% of the total bacterial number in the samples. The presence and number of E. coli did not correlate with any of physical and/or chemical characteristic of the drinking water (e.g., temperature, chlorine, or biodegradable organic matter concentration). We show here that E. coli is present in the biofilms of drinking water networks in Europe. Some of the cells are metabolically active but are often not detected due to limitations of traditionally used culture-based methods, indicating that biofilm should be considered as a reservoir that must be investigated further in order to evaluate the risk for human health.     

Effect of Escherichia coli morphogene bolA on biofilms

Biofilm physiology is established under a low growth rate. The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development. In order to verify this hypothesis, we tested the effect of bolA on biofilm formation. Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms.     

Formation of nonculturable Escherichia coli in drinking water

AIMS: To examine whether incubation of Escherichia coli in nondisinfected drinking water result in development of cells that are not detectable using standard procedures but maintain a potential for metabolic activity and cell division. METHODS AND RESULTS: Survival and detectability of four different E. coli strains were studied using drinking water microcosms and samples from contaminated drinking water wells. Recovery of E. coli was compared using different cultivation-dependent methods, fluorescence in situ hybridization (FISH) using specific oligonucleotide probes, direct viable counts (DVC), and by enumeration of gfp-tagged E. coli (green fluorescent protein, GFP). Two levels of stress responses were observed after incubation of E. coli in nondisinfected drinking water: (i) the presence of cells that were not detected using standard cultivation methods but could be cultivated after gentle resuscitation on nonselective nutrient-rich media, and (ii) the presence of cells that responded to nutrient addition but could only be detected by cultivation-independent methods (DVC, FISH and GFP). Collectively, the experiments demonstrated that incubation for 20-60 days in nondisinfected drinking water resulted in detection of only 0.7-5% of the initial E. coli population using standard cultivation methods, whereas 1-20% could be resuscitated to a culturable state, and 17-49% could be clearly detected using cultivation-independent methods. CONCLUSIONS: Resuscitation of stressed E. coli on nonselective nutrient-rich media increased cell counts in drinking water using both traditional (CFU), and cultivation-independent methods (DVC, FISH and GFP). The cultivation-independent methods resulted in detection of 10-20 times more E. coli than the traditional methods. The results indicate that a subpopulation of substrate-responsive but apparent nonculturable E. coli may develop in drinking water during long-term starvation survival. SIGNIFICANCE AND IMPACT OF THE STUDY: The existence of substrate-responsive but nonculturable cells should be considered when evaluating the survival potential of E. coli in nondisinfected drinking water.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4 degrees to 25 degrees C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 micron Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4 degrees C greater than 15 degrees C greater than 25 degrees C greater than 37 degrees C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R1drd-19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15 degrees C or of loss of plasmid function during starvation.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4° to 25°C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 μm Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4°C > 15°C > 25°C > 37°C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R 1 drd -19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15°C or of loss of plasmid function during starvation.     

Mycobacterium xenopi and drinking water biofilms

The ability of Mycobacterium xenopi to colonize an experimental drinking water distribution system (a Propella reactor) was investigated. M. xenopi was present in the biofilm within an hour following its introduction. After 9 weeks, it was always present in the outlet water (1 to 10 CFU 100 ml(-1)) and inside the biofilm (10(2) to 10(3) CFU cm(-2)). Biofilms may be considered reservoirs for the survival of M. xenopi.     

Global gene expression in Escherichia coli biofilms

It is now apparent that microorganisms undergo significant changes during the transition from planktonic to biofilm growth. These changes result in phenotypic adaptations that allow the formation of highly organized and structured sessile communities, which possess enhanced resistance to antimicrobial treatments and host immune defence responses. Escherichia coli has been used as a model organism to study the mechanisms of growth within adhered communities. In this study, we use DNA microarray technology to examine the global gene expression profile of E. coli during sessile growth compared with planktonic growth. Genes encoding proteins involved in adhesion (type 1 fimbriae) and, in particular, autoaggregation (Antigen 43) were highly expressed in the adhered population in a manner that is consistent with current models of sessile community development. Several novel gene clusters were induced upon the transition to biofilm growth, and these included genes expressed under oxygen-limiting conditions, genes encoding (putative) transport proteins, putative oxidoreductases and genes associated with enhanced heavy metal resistance. Of particular interest was the observation that many of the genes altered in expression have no current defined function. These genes, as well as those induced by stresses relevant to biofilm growth such as oxygen and nutrient limitation, may be important factors that trigger enhanced resistance mechanisms of sessile communities to antibiotics and hydrodynamic shear forces.     

Effect of Phosphorus on Survival of Escherichia coli in Drinking Water Biofilms

The effect of phosphorus addition on survival of Escherichia coli in an experimental drinking water distribution system was investigated. Higher phosphorus concentrations prolonged the survival of culturable E. coli in water and biofilms. Although phosphorus addition did not affect viable but not culturable (VBNC) E. coli in biofilms, these structures could act as a reservoir of VBNC forms of E. coli in drinking water distribution systems.     

Highly sensitive and specific detection of viable Escherichia coli in drinking water

A highly sensitive and specific assay method was developed for the detection of viable Escherichia coli as an indicator organism in water, using nucleic acid sequence-based amplification (NASBA) and electrochemiluminescence (ECL) analysis. Viable E. coli were identified via a 200-nt-long target sequence from mRNA (clpB) coding for a heat shock protein. In the detection assay, a heat shock was applied to the cells prior to disruption to induce the synthesis of clpB mRNA and the mRNA was extracted, purified, and finally amplified using NASBA. The amplified mRNA was quantified with an ECL detection system after hybridization with specific DNA probes. Several disruption methods were investigated to maximize total RNA extracted from viable cells. Optimization was also carried out regarding the design of NASBA primer pairs and detection probes, as well as reaction and detection conditions. Finally, the assay was tested regarding sensitivity and specificity. Analysis of samples revealed that as few as 40 E. coli cells/mL can be detected, with no false positive signals resulting from other microorganisms or nonviable E. coli cells. Also, it was shown that a quantification of E. coli cells was possible with our assay method.     

Effect of millimeter waves on survival of UVC-exposed Escherichia coli

Bacterial cells of the strain Escherichia coli K12 were exposed to millimeter electromagnetic waves (mm waves) with and without additional exposure to ultraviolet light λ = 254 nm (UVC). The mm waves were produced by a medical microwave generator emitting a 4-GHz-wide band around a 61 GHz center frequency and delivering an irradiation of 1 mW/cm² and a standard absorption rate (SAR) of 84 W/kg to the bacteria. Exposure to the mm waves alone for up to 30 minutes did not change the survival rate of bacteria. Exposure to mm waves followed by UVC irradiation also did not alter the number of surviving E. coli cells in comparison to UVC-treated controls. When mm waves were applied after the UVC exposure, a dose-dependent increase of up to 30% in the survival of E. coli was observed compared to UVC + sham-irradiated bacteria. Because sham controls and experimental samples were maintained under the same thermal conditions, the effect is not likely to be due to heating, although the possibility of nonuniform distribution of microwave heating in different layers of irradiated bacterial suspension cannot be ruled out. The mechanism for this effect appears to involve certain DNA repair systems that act as cellular targets for mm waves. © 1995 Wiley-Liss, Inc.     

Effect of oxygen on survival of faecal pollution indicators in drinking water

AIMS: The aim of this study was to determine the effect of oxygen on the survival of faecal pollution indicators including Escherichia coli in nondisinfected drinking water. METHODS AND RESULTS: Aerobic and anaerobic drinking water microcosms were inoculated with E. coli ATCC 25922 or raw sewage. Survival of E. coli was monitored by membrane filtration combined with cultivation on standard media, and by in situ hybridization with 16S rRNA-targeted fluorescent oligonucleotide probes. Anaerobic conditions significantly increased the survival of E. coli in drinking water compared with aerobic conditions. Escherichia coli ATCC 25922 showed a biphasic decrease in survival under aerobic conditions with an initial first-order decay rate of -0.11 day(-1) followed by a more rapid rate of -0.35 day(-1). In contrast, the first-order decay rate under anaerobic conditions was only -0.02 day(-1). After 35 days, <0.01% of the initial E. coli ATCC 25922 population remained detectable in aerobic microcosms compared with 48% in anaerobic microcosms. A poor survival was observed under aerobic conditions regardless of whether E. coli ATCC 25922 or sewage-derived E. coli was examined, and regardless of the detection method used (CFU or fluorescent in situ hybridization). Aerobic conditions in drinking water also appeared to decrease the survival of faecal enterococci, somatic coliphages and coliforms other than E. coli. CONCLUSIONS: The results indicate that oxygen is a major regulator of the survival of E. coli in nondisinfected drinking water. The results also suggest that faecal pollution indicators other than E. coli may persist longer in drinking water under anaerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The effect of oxygen should be considered when evaluating the survival potential of enteric pathogens in oligotrophic environments.     

Turbulence accelerates the growth of drinking water biofilms

Bioprocess and Biosystems Engineering - Biofilms are found at the inner surfaces of drinking water pipes and, therefore, it is essential to understand biofilm processes to control their formation....     

The effects of nutrients on the survival of Escherichia coli in lake water

Escherichia coli was shown to survive without decline in viable counts for at least 12 d in filtered-autoclaved lake water. In unfiltered lake water there was a rapid decline in the viable count of E. coli. The addition of synthetic sewage to filtered-autoclaved lake water led to an increase in the viable count of E. coli at 15 degrees C and 37 degrees C and to an increase in the survival time of the E. coli in unfiltered water. The addition of phosphate and carbon sources (glucose, glycerol, succinate, acetate and lactose) did not significantly increase the survival time of E. coli in unfiltered water over the controls. The addition of ammonium sulphate and some amino acids (as nitrogen sources) to the unfiltered lake water did lead to an increase in the survival times for E. coli and this increase was proportional to the concentration of the added nitrogen source.     

The effects of nutrients on the survival of Escherichia coli in lake water

Escherichia coli was shown to survive without decline in viable counts for at least 12 d in filtered-autoclaved lake water. In unfiltered lake water there was a rapid decline in the viable count of E. coli. The addition of synthetic sewage to filtered-autoclaved lake water led to an increase in the viable count of E. coli at 15°C and 37°C and to an increase in the survival time of the E. coli in unfiltered water. The addition of phosphate and carbon sources (glucose, glycerol, succinate, acetate and lactose) did not significantly increase the survival time of E. coli in unfiltered water over the controls. The addition of ammonium sulphate and some amino acids (as nitrogen sources) to the unfiltered lake water did lead to an increase in the survival times for E. coli and this increase was proportional to the concentration of the added nitrogen source.     

RNA biosensor for the rapid detection of viable Escherichia coli in drinking water

A highly sensitive and specific RNA biosensor was developed for the rapid detection of viable Escherichia coli as an indicator organism in water. The biosensor is coupled with protocols developed earlier for the extraction and amplification of mRNA molecules from E. coli [Anal. Biochem. 303 (2002) 186]. However, in contrast to earlier detection methods, the biosensor allows the rapid detection and quantification of E. coli mRNA in only 15-20 min. In addition, the biosensor is portable, inexpensive and very easy to use, which makes it an ideal detection system for field applications. Viable E. coli are identified and quantified via a 200 nt-long target sequence from mRNA (clpB) coding for a heat shock protein. For sample preparation, a heat shock is applied to the cells prior to disruption. Then, mRNA is extracted, purified and finally amplified using the isothermal amplification technique Nucleic acid sequence-based amplification (NASBA). The amplified RNA is then quantified with the biosensor. The biosensor is a membrane-based DNA/RNA hybridization system using liposome amplification. The various biosensor components such as DNA probe sequences and concentration, buffers, incubation times have been optimized, and using a synthetic target sequence, a detection limit of 5 fmol per sample was determined. An excellent correlation to a much more elaborate and expensive laboratory based detection system was demonstrated, which can detect as few as 40 E. coli cfu/ml. Finally, the assay was tested regarding its specificity; no false positive signals were obtained from other microorganisms or from nonviable E. coli cells.