Competitive polymerase chain reaction for quantification of nonculturable Enterococcus faecalis cells in lake water

Among the survival strategies developed by bacteria when faced with adverse environmental conditions, the viable but nonculturable (VNC) state has been described. In this state, bacteria are unable to form colonies but are still alive and capable of metabolic activity. The VNC state has been described in numerous Gram-negative species, but recently also in Enterococcus faecalis, a Gram-positive species which can be found in the environment. In this study we describe a competitive PCR (cPCR) protocol to detect and quantify a specific sequence of DNA from culturable and nonculturable E. faecalis cells present in water samples. The protocol was found to be specific and capable of detecting amounts of DNA up to 0.1 pg corresponding to approximately 2 cells ml(-1). Moreover, it allows an internal standard to be used to quantify the amount of specific DNA present in samples from different environments. The application of this cPCR method to water samples from Lake Garda enabled us to demonstrate the presence of nonculturable forms of E. faecalis in lake water and to quantify their DNA and the corresponding concentration of nonculturable cells.     

Detection of Campylobacter jejuni and Campylobacter coli in environmental waters by PCR enzyme-linked immunosorbent assay

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.     

Optimization of conditions for the polymerase chain reaction amplification of DNA from culturable and nonculturable cells of Vibrio vulnificus

Abstract: A series of 16 buffers, differing in pH and MgCl₂ concentration, were used to optimize the polymerase chain reaction (PCR) amplification of a 388 bp region of the hemolysin / cytolysin gene from cells of Vibrio vulnificus present in both the culturable and nonculturable states. Both the opaque and translucent morphotypes were examined. Using whole cell lysates, we were able to obtain amplification of DNA from as few as 28.5 cells present in the viable but nonculturable state. With one exception, all buffers that produced amplification using culturable cells also produced amplification using nonculturable cells. However, regardless of the buffer employed, 100 times more nonculturable cells than culturable cells were required to obtain a PCR product. Our data suggest that caution should be exercised when employing PCR optimized against culturable cells when this method is employed for the detection of nonculturable cells.     

Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.     

Use of the polymerase chain reaction in detection of culturable and nonculturable Vibrio vulnificus cells.

Vibrio vulnificus is a human pathogen associated with consumption of raw oysters. During the colder months the organism apparently enters a viable but nonculturable state and thus cannot be cultured by ordinary bacteriological methods. For this reason, another means of detecting this bacterium is necessary. In the present study we utilized the polymerase chain reaction (PCR) to detect V. vulnificus DNA, thus eliminating the problem of nonculturability. DNA from both culturable and nonculturable cells of V. vulnificus was amplified by PCR with primers flanking a 340-bp fragment of the cytotoxin-hemolysin gene. As little as 72 pg of DNA from culturable cells and 31 ng of DNA from nonculturable cells could be detected. Fifty cycles of a two-step reaction (30 s [each] at 94 and 65 degrees C) were found to be optimal as well as more time efficient than the three-step PCR. The total procedure from the point of DNA extraction to observation on a gel required less than 8 h. Possible reasons for the difficulties encountered in amplifying DNA from nonculturable cells, e.g., gene rearrangement or loss of the hemolysin gene, are discussed.     

Specific detection of Arcobacter spp. in estuarine waters of Southern Italy by PCR and fluorescent in situ hybridization

Aim:  To evaluate the reliability of culture-independent methods in comparison with culture-dependent ones for the detection of Arcobacter spp. in estuarine waters of Southern Italy.
Methods and Results:  PCR and fluorescent in situ hybridization (FISH) procedures were used to detect arcobacters directly in water samples and after enrichment cultures. The samples totally were positive by molecular methods (PCR and FISH) but only 75% were culture positive, confirming the limitation of these latter to detect Arcobacter spp. in natural samples. Culturable arcobacters were retrieved in all times except in July, and isolated species were ascribed only to Arcobacter cryaerophilus .
Conclusions:  Culturable and nonculturable forms of Arcobacter in the estuarine environment were present. PCR assays were more sensitive than traditional culture in detecting Arcobacter butzleri and A. cryaerophilus . FISH comparatively to PCR technique may provide information about cell morphology and viability of single cells.
Significance and Impact of the Study:  Our investigation indicates the existence of an environmental reservoir of potential pathogenic arcobacters in an estuarine Italian area, which may survive under a viable but not culturable state.     

Detection of culturable and nonculturable Legionella species from hot water systems of public buildings in Japan

Aims: To investigate the prevalence of culturable and nonculturable Legionella species in hot water systems of public buildings in Japan and assess the risk factors associated with Legionella contamination in hot water systems. Methods and Results: Legionella species were detected by conventional culture and molecular methods in 130 water samples collected from 40 buildings. A total of 26 (20·0%) water samples from 17 (42·5%) buildings were positive by culture, qualitative PCR or both methods: Legionella pneumophila and Leg. anisa were detected in four samples by a culture method, whereas 23 samples were positive by qualitative PCR, with the presence of various Legionella species confirmed by sequencing. Of these 23 samples, bacterial counts were quantifiable in 21 by real‐time PCR (from 1·7 × 10⁵ to 2·6 × 10¹¹ cells per litre). Phylogenetic analysis of amplified partial 16S rRNA gene showed close relations to various species of Legionella, including Leg. anisa and Leg. micdadei, all of which have been associated with respiratory diseases or increased antibody titres in human sera. Assessment of risk factors showed that turbidity, free chlorine concentration, iron concentration and heterotrophic plate count (HPC) were significantly associated with Legionella contamination (P < 0·05). Conclusions: Contamination of hot water systems of public buildings with culturable and nonculturable Legionella species may be a potential risk factor for Legionella infection in Japan. Adequate levels of chlorine, low levels of iron and HPC are important maintenance measures in the reduction of Legionella contamination in hot water systems. Significance and Impact of the Study: More than 40% of hot water systems in the Japanese public buildings examined were contaminated by not only culturable Leg. pneumophila and Leg. anisa but also by nonculturable pathogenic species. To our knowledge, this is the first report of both culturable and nonculturable Legionella contamination in hot water systems of public buildings in Japan.     

Detection of Campylobacter jejuni and Campylobacter coli in Environmental Waters by PCR Enzyme-Linked Immunosorbent Assay

A PCR enzyme-linked immunosorbent assay (ELISA) assay was applied to the detection of Campylobacter jejuni and Campylobacter coli in environmental water samples after enrichment culture. Bacterial cells were concentrated from 69 environmental water samples by using filtration, and the filtrates were cultured in Campylobacter blood-free broth. After enrichment culture, DNA was extracted from the samples by using a rapid-boiling method, and the DNA extracts were used as a template in a PCR ELISA assay. A total of 51 samples were positive by either PCR ELISA or culture; of these, 43 were found to be positive by PCR ELISA and 43 were found to be positive by culture. Overall, including positive and negative results, 59 samples were concordant in both methods. Several samples were positive in the PCR ELISA assay but were culture negative; therefore, this assay may be able to detect sublethally damaged or viable nonculturable forms of campylobacters. The method is rapid and sensitive, and it significantly reduces the time needed for the detection of these important pathogens by 2 to 3 days.     

Examination of recovery in vitro and in vivo of nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7 (strains ATCC 43895 and FO46) became nonculturable in sterile, distilled, deionized water or after exposure to chlorine. Recovery of nonculturable E. coli O157:H7 was examined by in vitro and in vivo methods. The decline in culturability of starved E. coli O157:H7 was measured by plate count on rich medium. Recovery in vitro of nonculturable cells was conducted with media amended with catalase or sodium pyruvate; however, there was no apparent increase over culturable cell counts on amended versus nonamended media. Although nonculturable E. coli O157:H7 did not recover under in vitro conditions, a mouse model was used to determine if in vivo conditions would provide sufficient conditions for recovery of nonculturable E. coli O157:H7. In separate studies, mice were orally challenged with starvation-induced nonculturable cells (FO46) or chlorine-induced nonculturable cells (43895 and FO46). Passage through the mouse gastrointestinal tract had no effect on recovery of nonculturable (starvation or chlorine induced) E. coli O157:H7 (43895 or FO46), based on analysis of fecal samples. Mouse kidneys were assayed for the presence of Shiga toxin using the Vero cell assay. Differences in cytotoxicity towards Vero cells from kidney samples of mice receiving nonculturable cells and control mice were not significant, suggesting a loss of virulence.     

Escherichia coli detection using mTEC agar and fluorescent antibody direct viable counting on coastal recreational water samples

Aims:  Escherichia coli is the faecal indicator species recommended by the US Environmental Protection Agency (USEPA) for monitoring fresh recreational water. Viable but nonculturable (VBNC) E. coli are living cells that are dormant and not culturable using standard microbiological cultivation methods. This study reports a comparison between the mTEC culture method recommended by USEPA for E. coli enumeration and a fluorescent antibody-direct viable count (FA-DVC) method to visualize living E. coli cells with a microscope.
Methods and Results:  Escherichia coli , faecal coliforms and Enterococcus were detected using standard methods recommended by the USEPA. VBNC E. coli was visualized with FA-DVC. Results were analysed with standard statistical methods (Pearson correlation; paired-sample t -test). Significantly higher numbers of E. coli were detected using the FA-DVC method than using the mTEC method. Escherichia coli results were also compared with faecal coliform (mFC broth) and Enterococcus (mEI agar) counts in the same samples.
Conclusions:  The results of this comparative study demonstrate that E. coli can be present in higher numbers than what are detected with standard culture methods.
Significance and Impact of the Study:  This study re-emphasizes the need for a rapid, accurate and precise method for detecting health risks to humans who use recreational waters.     

Efficacy of filter types for detecting Campylobacter jejuni and Campylobacter coli in environmental water samples by polymerase chain reaction.

A previously developed polymerase chain reaction (PCR) amplification of a target region in the flaA Campylobacter flagellin gene was evaluated and adapted for use with environmental water samples. The ability to detect Campylobacter jejuni or Campylobacter coli in seeded water samples was tested with various filters after concentration and freeze-thaw lysis of the bacterial cells. A nonradioactive probe for the amplified flagellin gene fragment detected as little as 1 to 10 fg of genomic DNA and as few as 10 to 100 viable C. jejuni cells per 100 ml of water filtered onto Fluoropore (Millipore Corp.) filters. No amplification was obtained with cellulose acetate filters, most likely because of binding of the DNA to the filter. Concentration and lysis of target cells on Fluoropore and Durapore (Millipore Corp.) filters allowed PCR to be performed in the same reaction tube without removing the filters. This methodology was then adapted for use with environmental water samples. The water supply to a broiler chicken production farm was suspected as the source of C. jejuni known to be endemic in grow-out flocks at the farm, despite the inability to culture the organisms by standard methods. The filtration-PCR method detected Campylobacter DNA in more than half of the farm water samples examined. Amplified campylobacter DNA was not detected in small volumes of regional surface water samples collected on a single occasion in February. The filtration-PCR amplification method provided a basis for detection of C. jejuni and C. coli in environmental waters with a high degree of specificity and sensitivity.     

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.     

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Survival of Aeromonas salmonicida in lake water.

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Survival of Aeromonas salmonicida in lake water

The survival of Aeromonas salmonicida subsp. salmonicida in lake water was investigated by using a variety of techniques. They included acridine orange epifluorescence, respiration, cell culture, cell revival, flow cytometry, plasmid maintenance, and membrane fatty acid analysis. During a 21-day study, A. salmonicida became nonculturable in sterile lake water samples. Flow cytometry and direct microscopy indicated that cells were present. Although the nonculturable cells could not be revived, the recovery method did indicate that the presence of low numbers of culturable cells within samples could produce misleading results. Plasmid DNA, genomic DNA, and RNA were maintained in the nonculturable cells; in addition, changes in the fatty acid profiles were also detected. Although viability could not be proven, it was shown that the morphological integrity of nonculturable cells was maintained.     

Inhibition of adhesion of food-borne pathogens to Caco-2 cells by Lactobacillus strains

AIMS: To select adhesive strains among strains of Lactobacillus and to apply them to inhibit adhesion of food-borne pathogens. METHODS AND RESULTS: Twelve Lactobacillus strains (10 from intestine) were examined for adhesion using Caco-2 cell cultures. The two most adhesive strains, Lactobacillus crispatus JCM 8779 and Lact. reuteri JCM 1081, were used to test antiadhesion activity against enterotoxigenic Escherichia coli, Salmonella typhimurium and Enterococcus faecalis strains. Adhesion of the pathogens was inhibited by both Lactobacillus strains. Adhesion of Ent. faecalis was especially strongly inhibited by JCM 8779. Although antimicrobial activity was not detected in the culture supernatant fluid by agar well diffusion assay, the supernatant fluid obtained from the harvested JCM 8779 cell suspension showed bactericidal activity against Ent. faecalis. CONCLUSION: The strong antiadhesion activity of JCM 8779 against Ent. faecalis appears to be due to the combined effect of both bactericidal activity and competition for attachment site. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that Lact. crispatus produces a bactericidal substance.     

Investigation of the first events leading to loss of culturability during Escherichia coli starvation: future nonculturable bacteria form a subpopulation

In previous experiments we were able to separate, using a nondestructive separation technique, culturable and nonculturable bacteria, from a Luria-Bertani (LB) medium culture of Escherichia coli incubated for 48 h. We observed in the nonculturable bacterial population an increase in oxidative damage and up-induction of most defenses against reactive oxygen species (ROS), along with a decrease in cytoplasmic superoxide dismutases. In this study, using the same separation technique, we separated into two subpopulations a 10-h LB medium culture containing only culturable bacteria. For the first time, we succeeded in associating physical separation with physiological differences. Although the levels of defense against ROS (RpoS, RpoH, OxyR, and SoxRS regulons) and oxidative damage (carbonyl contents) were apparently the same, we found that bacteria in one subpopulation were more sensitive to LB medium starvation and to various stresses, such as phosphate buffer starvation, heat shock, and hydrogen peroxide exposure. Based on these results, we suggest that these physiological differences reflect uncharacterized bacterial modifications which do not directly involve defenses against ROS.     

Induction of viable but nonculturable Escherichia coli O157:H7 in the phyllosphere of lettuce: a food safety risk factor

Escherichia coli O157:H7 continues to be an important human pathogen and has been increasingly linked to food-borne illness associated with fresh produce, particularly leafy greens. The aim of this work was to investigate the fate of E. coli O157:H7 on the phyllosphere of lettuce under low temperature and to evaluate the potential hazard of viable but nonculturable (VBNC) cells induced under such stressful conditions. First, we studied the survival of six bacterial strains following prolonged storage in water at low temperature (4°C) and selected two strains with different nonculturable responses for the construction of E. coli O157:H7 Tn7gfp transformants in order to quantitatively assess the occurrence of human pathogens on the plant surface. Under a suboptimal growth temperature (16°C), both E. coli O157:H7 strains maintained culturability on lettuce leaves, but under more stressful conditions (8°C), the bacterial populations evolved toward the VBNC state. The strain-dependent nonculturable response was more evident in the experiments with different inoculum doses (10(9) and 10(6) E. coli O157:H7 bacteria per g of leaf) when strain BRMSID 188 lost culturability after 15 days and strain ATCC 43895 lost culturability within 7 days, regardless of the inoculum dose. However, the number of cells entering the VBNC state in high-cell-density inoculum (approximately 55%) was lower than in low-cell-density inoculum (approximately 70%). We recorded the presence of verotoxin for 3 days in samples that contained a VBNC population of 4 to 5 log(10) cells but did not detect culturable cells. These findings indicate that E. coli O157:H7 VBNC cells are induced on lettuce plants, and this may have implications regarding food safety.     

Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state

The viabilities of five strains of Vibrio vulnificus were evaluated during the storage of the organisms in sterile seawater at 5 degrees C. The number of CFU was measured by plate count methods on rich media. The total cell numbers were determined by direct microscopic count methods. The titer of CFU declined logarithmically to undetectable levels over a period of 2 to 3 weeks, while the total cell numbers were unchanged. Midway through each study, higher culturable cell counts began to be observed on plates containing catalase or sodium pyruvate; during the latter stages of the study, the plate counts on such media were up to 1,000-fold higher than those on unsupplemented plates. Because autoclaving is known to generate hydrogen peroxide in rich media, and because catalase and sodium pyruvate are known to eliminate hydrogen peroxide, it appears that the conditions of the experiments led to the selection of a hydrogen peroxide-sensitive culturable cell subpopulation. At the time of the final stage of the decline in viability of each culture, hydrogen peroxide-sensitive cells were the only culturable cells present. Warming samples of the cultures to room temperature led to the growth of these residual culturable cells, utilizing nutrients provided by the nonculturable cells. The cells that grew recovered hydrogen peroxide resistance. When mixtures of culturable and nonculturable cells were diluted to the point where only nonculturable cells were present, or when the hydrogen peroxide-sensitive culturable cells had declined to undetectable levels, warming had no effect; no culturable cells were recovered. Warming has been reported to "resuscitate" nonculturable cells. Recognition of the existence of hydrogen peroxide-sensitive culturable cell populations, as well as their ability to grow to high levels in the warmed seawater microcosms, leads instead to the conclusion that while warming permits culturable cells to grow, it has no effect on nonculturable cells.     

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.